CN1809536A - Inhibitors of Akt activity - Google Patents

Abstract

The present invention is directed to compounds which contain a substituted pyridine moiety which inhibit the activity of Akt, a serine/threonine protein kinase. The invention is further directed to chemotherapeutic compositions containing the compounds of this invention and methods for treating cancer comprising administration of the compounds of the invention.

Description

Akt activity inhibitor

Background of the Invention

The present invention relates to substituted pyridine-containing compounds that are inhibitors of the activity of one or more isoforms of the serine/threonine kinase Akt (also known as PKB; hereinafter abbreviated as "Akt"). The invention also relates to pharmaceutical compositions comprising such compounds and methods of using the compounds of the invention for the treatment of cancer.

Apoptosis (programmed cell death) plays a major role in the development of the embryo and in the pathogenesis of various diseases such as degenerative neuronal diseases, cardiovascular diseases and cancer. Recent studies have identified various pro-apoptotic and anti-apoptotic gene products involved in the regulation or execution of programmed cell death. Expression of anti-apoptotic genes such as Bcl2 or Bcl-x _L can inhibit apoptosis induced by various stimuli. On the other hand, expression of pro-apoptotic genes such as Bax or Bad leads to programmed cell death (Aams et al., Science, 281:1322-1326 (1998)). The execution of programmed cell death is mediated by caspase-1-related proteases, including caspase-3, caspase-7, caspase-8, and caspase-9, etc. (Thornberry et al., Science, 281: 1312-1316 (1998)).

The phosphatidylinositol 3'-OH kinase (PI3K)/Akt pathway appears to be important for regulating cell survival/cell death (Kulik et al., Mol. Cell. Biol. 17:1595-1606 (1997); Franke et al., Cell, 88 : 435-437 (1997); Kauffmann-Zeh et al., Nature 385: 544-548 (1997) Hemmings Science, 275: 628-630 (1997); Dudek et al., Science, 275: 661-665 (1997)). Survival factors, such as platelet-derived growth factor (PDGF), nerve growth factor (NGF), and insulin-like growth factor-1 (IGF-1), can promote cell survival under various conditions by inducing the activity of PI3K (Kulik et al. 1997, Hemmings 1997). Activation of PI3K results in the production of phosphatidylinositol (3,4,5)-triphosphate (PtdIns(3,4,5)-P3), which in turn binds to the pleckstrand-containing substrate protein homology (PH)- Serine/threonine kinase Akt of domain and promote its activation (Franke et al., Cell, 81:727-736 (1995); Hemmings Science, 277:534 (1997); Downward, Curr.Opin.Cell Biol.10: 262-267 (1998), Alessi et al., EMBO J. 15:6541-6551 (1996)). Specific inhibitors of PI3K or dominant-negative Akt mutants completely abolish the pro-survival activity of these growth factors or cytokines. It was previously revealed that PI3K inhibitors (LY294002 or wortmannin) block activation of Akt by upstream kinases. Furthermore, introduction of constitutively active PI3K or Akt mutants can promote cell survival under conditions that would normally lead to apoptosis (Kulik et al., 1997, Dudek et al., 1997).

Three members of the Akt subfamily of second messenger-regulated serine/threonine protein kinases have been identified and named Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ (hereinafter referred to as “Akt1”, “Akt2” and “ Akt3"). These isoforms are homologous, especially in the region encoding the catalytic domain. Akt is activated through phosphorylation that occurs in response to PI3K signaling. PI3K phosphorylates inositol phospholipids on the membrane to produce the second messengers phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate, the latter two of which have been shown to bind the PH domain of Akt. Existing models of Akt activation propose that the enzyme is recruited to the membrane by 3′-phosphorylated phosphoinositides, where phosphorylation of Akt regulatory sites by upstream kinases occurs (B.A. Hemmings, Science 275:628-630 (1997); B.A. Hemmings, Science 276:534 (1997); J. Downward, Science 279:673-674 (1998)).

Phosphorylation of Akt1 occurs at two regulatory sites: Thr ³⁰⁸ in the activation loop of the catalytic domain and Ser ⁴⁷³ near the carboxyl terminus (DRAlessi et al., EMBO J.15:6541-6551 (1996) and R.Meier et al., J. Biol. Chem. 272:30491-30497 (1997)). Equivalent phosphorylation regulatory sites also occur in Akt2 and Akt3. The upstream kinase that phosphorylates Akt at the activation loop site has been cloned and named 3'-phosphoinositide-dependent protein kinase (PDK1). PDK1 phosphorylates not only Akt, but also p70 ribosomal S6 kinase, p90RSK, serum- and glucocorticoid-regulated kinase (SGK), and protein kinase C. Upstream kinases that phosphorylate the Akt/PKB regulatory site near the carboxyl terminus have not been identified, but recent reports implicate integrin-coupled kinase (ILK-1, a serine/threonine protein kinase) or autophosphorylation role.

Analysis of Akt levels in human tumors shows that Akt2 is expressed in a large number of ovarian cancers (J.Q.Cheng et al., Proc. . Natl. Acad. Sci. U.S.A. 93:3636-3641 (1996)) overexpression. Similarly, Akt3 was found to be overexpressed in breast and prostate cancer cell lines (Nakatani et al., J. Biol. Chem. 274:21528-21532 (1999).

The tumor suppressor protein PTEN, a protein and lipid phosphatase that specifically removes the 3′-phosphate of PtdIns(3,4,5)-P3, is a negative regulator of the PI3K/Akt pathway (Li et al., Science 275:1943 -1947 (1997), Stambolic et al., Cell 95:29-39 (1998), Sun et al., Proc. Natl. Acad. Sci. U.S.A. 96:6199-6204 (1999)). Germline mutations in PTEN cause human cancer syndromes such as Cowden's disease (Liaw et al., Nature Genetics 16:64-67 (1997)). PTEN is absent in most human tumors, and tumor cell lines without functional PTEN show elevated levels of activated Akt (Li et al., supra, Guldberg et al., Cancer Research 57:3660-3663 (1997), Risinger et al., Cancer Research 57: 4736-4738 (1997)).

These observations above suggest that the PI3K/Akt pathway plays an important role in regulating cell survival or apoptosis in tumorigenesis.

Inhibition of Akt activation and activity can be achieved by inhibiting PI3K with inhibitors such as LY294002 or wortmannin. However, inhibition of PI3K may indiscriminately affect all three Akt isozymes as well as other PtdIns(3,4,5)-P3-dependent PH domain-containing signaling molecules such as the Tec family of tyrosine kinases. Furthermore, Akt has been revealed to be activated by PI3K-independent growth signals.

In addition, Atk activity can be inhibited by blocking the activity of the upstream kinase PDK1. No specific PDK1 inhibitors have been disclosed. Furthermore, inhibition of PDK1 results in inhibition of various protein kinases whose activity is dependent on PDK1, such as atypical PKC isoforms, SGK and S6 kinases (Williams et al., Curr. Biol. 10:439-448 (2000).

It is an object of the present invention to provide novel compounds which are inhibitors of Akt.

Yet another object of the present invention is to provide pharmaceutical compositions comprising novel compounds that are Akt inhibitors.

Still another object of the present invention is to provide a method of treating cancer comprising administering such an inhibitor of Akt activity.

Invention Overview

The present invention provides compounds comprising substituted pyridines which inhibit Akt activity. In particular, the disclosed compounds are capable of selectively inhibiting one or both Akt isoforms. The invention also provides compositions comprising such inhibitory compounds, and methods of inhibiting Akt activity by administering the compounds to a patient in need of treatment for cancer.

                      Invention Details

The compounds of the invention are useful for inhibiting the activity of the serine/threonine kinase Akt. In the first embodiment of the present invention, the Akt activity inhibitor is a compound shown in formula A or its pharmaceutically acceptable salt or stereoisomer:

in:

a is 0 or 1; b is 0 or 1; m is 0, 1 or 2; n is 0, 1, 2 or 3; p is 0, 1 or 2; q is 0, 1, 2 or 3; r is 0 or 1; s is 0 or 1; t is 2, 3, 4, 5 or 6;

R ¹ is independently selected from 1) (C=O) _a O _b C ₁ -C ₁₀ alkyl, 2) (C=O) _a O _b aryl, 3) C ₂ -C ₁₀ alkenyl, 4) C ₂ -C ₁₀ alkynyl, 5) (C=O) _a O _b heterocyclyl, 6) (C=O) _a O _b C ₃ -C ₈ cycloalkyl, 7) CO ₂ H, 8) halogen, 9 ) CN, 10) OH, 11) O _b C ₁ -C ₆ perfluoroalkyl, 12) O _a (C=O) _b NR ⁶ R ⁷ , 13) NR ^c (C=O) NR ⁶ R ⁷ , 14) S(O) _m R ^a , 15) S(O) ₂ NR ⁶ R ⁷ , 16) NR ^c S(O) _m R ^a , 17) oxo, 18) CHO, 19) NO ₂ , 20) NR ^c (C=O)O _b R ^a , 21)O(C=O)O _b C ₁ -C ₁₀ alkyl, 22)O(C=O)O _b C ₃ -C ₈ cycloalkyl, 23 )O(C=O)O _b aryl, 24)O(C=O)O _b -heterocycle and 25)O _a -P=O(OH) ₂ , the alkyl, aryl, alkenyl, Alkynyl, heterocyclyl and cycloalkyl are optionally substituted by one or more substituents selected from ^Rz ;

R ² is independently selected from 1) (C=O) _a O _b C ₁ -C ₁₀ alkyl, 2) (C=O) _a O _b aryl, 3) C ₂ -C ₁₀ alkenyl, 4) C ₂ -C ₁₀ alkynyl, 5) (C=O) _a O _b heterocyclyl, 6) (C=O) _a O _b C ₃ -C ₈ cycloalkyl, 7) CO ₂ H, 8) halogen, 9 ) CN, 10) OH, 11) O _b C ₁ -C ₆ perfluoroalkyl, 12) O _a (C=O) _b NR ⁶ R ⁷ , 13) NR ^c (C=O) NR ⁶ R ⁷ , 14) S(O) _m R ^a , 15) S(O) ₂ NR ⁶ R ⁷ , 16) NR ^c S(O) _m R ^a , 17) CHO, 18) NO ₂ , 19) NR ^c (C= O)O _b R ^a , 20)O(C=O) _Ob C ₁ -C ₁₀ alkyl, 21)O(C=O)O _b C ₃ -C _8cycloalkyl , 22)O(C= O)O _b aryl, 23) O(C=O)O _b -heterocycle and 24)O _a -P=O(OH) ₂ , the alkyl, aryl, alkenyl, alkynyl, heterocycle and cycloalkyl are optionally substituted by one, two or three substituents selected from R ^z ;

R ³ and R ⁴ are independently selected from H, C ₁ -C ₆ -alkyl and C ₁ -C ₆ -perfluoroalkyl, or R ³ and R ⁴ are combined to form -(CH ₂ ) _t -, one of which A carbon atom is optionally replaced by a moiety selected from O, S(O) _m , -N( ^Rb )C(O)- and -N( ^CORa )-;

R ⁵ is independently selected from 1) (C=O) _a O _b C ₁ -C ₁₀ alkyl, 2) (C=O) _a O _b aryl, 3) C ₂ -C ₁₀ alkenyl, 4) C ₂ -C ₁₀ alkynyl, 5) (C=O) _a O _b heterocyclyl, 6) (C=O) _a O _b C ₃ -C ₈ cycloalkyl, 7) CO ₂ H, 8) halogen, 9 ) CN, 10) OH, 11) O _b C ₁ -C ₆ perfluoroalkyl, 12) O _a (C=O) _b NR ⁶ R ⁷ , 13) NR ^c (C=O) NR ⁶ R ⁷ , 14) S(O) _m R ^a , 15) S(O) ₂ NR ⁶ R ⁷ , 16) NR ^c S(O) _m R ^a , 17) oxo, 18) CHO, 19) NO ₂ , 20) O(C=O)O _b C ₁ -C ₁₀ alkyl, 21)O(C=O)O _b C ₃ -C ₈ cycloalkyl and 22)O _a -P=O(OH) ₂ , the Alkyl, aryl, alkenyl, alkynyl, heterocyclyl and cycloalkyl are optionally substituted by one or more substituents selected from ^R ;

R ⁶ and R ⁷ are independently selected from 1) H, 2) (C=O)O _b R ^a , 3) C ₁ -C ₁₀ alkyl, 4) aryl, 5) C ₂ -C ₁₀ alkenyl, 6 ) C ₂ -C ₁₀ alkynyl, 7) heterocyclyl, 8) C ₃ -C ₈ cycloalkyl, 9) SO ₂ R ^a , 10) (C=O)NR ^b ₂ , 11) OH and 12) O _a -P=O(OH) ₂ , the alkyl, cycloalkyl, aryl, heterocyclyl, alkenyl and alkynyl are optionally substituted by one or more substituents selected from R ^z ;

R ^z is selected from 1) (C=O) _r O _s (C ₁ -C ₁₀ ) alkyl, 2) O _r (C ₁ -C ₃ ) perfluoroalkyl, 3) (C ₀ -C ₆ ) alkylene Alkyl-S(O) _m R ^a , 4) oxo, 5) OH, 6) halogen, 7) CN, 8) (C=O) _r O _s (C ₂ -C ₁₀ ) alkenyl, 9) (C=O) _rOs ( _C2 - _C10 )alkynyl, 10)(C= _O ) _rOs ( _C3 - _C6 ) _cycloalkyl , 11)(C=O) _{rOs (} C ₀ -C ₆ )alkylene-aryl, 12)(C ₌ O) _rOs (C ₀ -C ₆ )alkylene-heterocyclyl, ₁₃ )(C=O) _rOs (C ₀ -C ₆ )alkylene-N(R ^b ) ₂ , 14)C(O)R ^a , 15)(C ₀ -C ₆ )alkylene-CO ₂ R ^a , 16)C(O)H , 17) (C ₀ -C ₆ ) alkylene-CO ₂ H, 18) C(O)N(R ^b ) ₂ , 19) S(O) _m R ^a , 20) S(O) ₂ N( R ^b ) ₂ , 21)NR ^c (C=O)O _b R ^a , 22)O(C=O)O _b C ₁ -C ₁₀ alkyl, 23)O(C=O)O _b C ₃ - C ₈ cycloalkyl, 24)O(C=O)O _b aryl, 25)O(C=O)O _b -heterocycle and 26)O _a -P=O(OH) ₂ , the alkyl , alkenyl, alkynyl, cycloalkyl, aryl and heterocyclyl are optionally replaced by up to three selected from R ^b , OH, (C ₁ -C ₆ )alkoxy, halogen, CO ₂ H, CN, O Substituents of (C=O)C ₁ -C ₆ alkyl, oxo, N(R ^b ) ₂ and O _a -P=O(OH) ₂ ;

R ^a is: substituted or unsubstituted (C ₁ -C ₆ ) alkyl, substituted or unsubstituted (C ₂ -C ₆ ) alkenyl, substituted or unsubstituted (C ₂ -C ₆ ) alkynyl, substituted or unsubstituted (C ₃ -C ₆ ) cycloalkyl, substituted or unsubstituted aryl, (C ₁ -C ₆ ) perfluoroalkyl, 2,2,2-trifluoroethyl or substituted or unsubstituted the heterocyclic group;

R ^b is: H, (C ₁ -C ₆ )alkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted heterocyclyl, (C ₃ -C ₆ )cycloalkane radical, (C=O)OC ₁ -C ₆ alkyl, (C=O)C ₁ -C ₆ alkyl or S(O) ₂ R ^a ;

R ^c is selected from 1) H, 2) C ₁ -C ₁₀ alkyl, 3) aryl, 4) C ₂ -C ₁₀ alkenyl, 5) C ₂ -C ₁₀ alkynyl, 6) heterocyclyl, 7 ) C ₃ -C ₈ cycloalkyl and 8) C ₁ -C ₆ perfluoroalkyl, said alkyl, cycloalkyl, aryl, heterocyclyl, alkenyl and alkynyl are optionally replaced by one or more Substituents selected from ^Rz are substituted.

The second embodiment of the present invention is a compound represented by formula B or a pharmaceutically acceptable salt or stereoisomer thereof:

in:

Rz ^' is selected from the group consisting of: alkyl, cycloalkyl, aryl, and heterocyclyl optionally substituted with 1-3 Rz ^'s ; all other substituents and variables are as defined in the first embodiment.

The third embodiment of the present invention is a compound represented by formula C or a pharmaceutically acceptable salt or stereoisomer thereof:

in:

R ⁶ is selected from: H and (C ₁ -C ₆ )alkyl; all other substituents and variables are as defined in the second embodiment.

Specific compounds of the invention include:

5-phenyl-6-[4-({[4-(1,2,3-thiadiazol-4-yl)benzyl]amino}methyl)phenyl]nicotinonitrile;

5-Phenyl-6-[4-({[(1S,2R)-2-phenylcyclopropyl]amino}methyl)phenyl]nicotinonitrile;

6-(4-{[(3,4-difluorobenzyl)amino]methyl}phenyl)-5-phenylnicotinonitrile;

6-[4-({[2-(3-fluorophenyl)ethyl]amino}methyl)phenyl]-5-phenylnicotinonitrile;

6-[4-({[2-(4-fluorophenyl)ethyl]amino}methyl)phenyl]-5-phenylnicotinonitrile;

5-phenyl-6-[4-({[(4-phenylmorpholin-2-yl)methyl]amino}methyl)phenyl]nicotinonitrile;

6-[4-({[(4-Benzylmorpholin-2-yl)methyl]amino}methyl)phenyl]-5-phenylnicotinonitrile;

6-[4-({Methyl[(1-phenyl-1H-pyrazol-4-yl)methyl]amino}methyl)phenyl]-5-phenylnicotinonitrile;

N-[2-(1-methylpyrrolidin-2-yl)ethyl]-N-{4-[3-phenyl-5-(1H-tetrazol-5-yl)pyridin-2-yl] Benzyl}amine;

(1-6)1-{4-[3-Phenyl-5-(1H-tetrazol-5-yl)pyridin-2-yl]phenyl}-N-[4-(1,2,3- Thiadiazol-4-yl)benzyl]methanamine;

N-(3,4-difluorobenzyl)-N-{4-[3-phenyl-5-(1H-tetrazol-5-yl)pyridin-2-yl]benzyl}amine;

2-Chloro-5-phenyl-6-[4-({[4-(1,2,3-thiadiazol-4-yl)benzyl]amino}methyl)phenyl]nicotinonitrile; (2 -6);

1-(2-aminophenyl)-3-({4-[5-(5-amino-1,3,4-thiadiazol-2-yl)-3-phenylpyridin-2-yl]benzyl Base}amino)propan-1-one (3-5);

3-({4-[5-cyano-3-phenylpyridin-2-yl]benzyl}amino)-1-phenylpropan-1-one;

3-({4-[5-(5-amino-1,3,4-thiadiazol-2-yl)-3-phenylpyridin-2-yl]benzyl}amino)-1-phenylpropane -1-one;

Or their pharmaceutically acceptable salts or stereoisomers.

Specific TFA salts of compounds of the invention include:

5-phenyl-6-[4-({[4-(1,2,3-thiadiazol-4-yl)benzyl]amino}methyl)phenyl]nicotinonitrile;

5-Phenyl-6-[4-({[(1S,2R)-2-phenylcyclopropyl]amino}methyl)phenyl]nicotinonitrile;

6-(4-{[(3,4-difluorobenzyl)amino]methyl}phenyl)-5-phenylnicotinonitrile;

6-[4-({[2-(3-fluorophenyl)ethyl]amino}methyl)phenyl]-5-phenylnicotinonitrile;

6-[4-({[2-(4-fluorophenyl)ethyl]amino}methyl)phenyl]-5-phenylnicotinonitrile;

5-phenyl-6-[4-({[(4-phenylmorpholin-2-yl)methyl]amino}methyl)phenyl]nicotinonitrile;

6-[4-({[(4-Benzylmorpholin-2-yl)methyl]amino}methyl)phenyl]-5-phenylnicotinonitrile;

6-[4-({Methyl[(1-phenyl-1H-pyrazol-4-yl)methyl]amino}methyl)phenyl]-5-phenylnicotinonitrile;

N-[2-(1-methylpyrrolidin-2-yl)ethyl]-N-{4-[3-phenyl-5-(1H-tetrazol-5-yl)pyridin-2-yl] Benzyl}amine;

1-{4-[3-phenyl-5-(1H-tetrazol-5-yl)pyridin-2-yl]phenyl}-N-[4-(1,2,3-thiadiazole-4 -yl)benzyl]methylamine;

N-(3,4-difluorobenzyl)-N-{4-[3-phenyl-5-(1H-tetrazol-5-yl)pyridin-2-yl]benzyl}amine;

1-(2-aminophenyl)-3-({4-[5-(5-amino-1,3,4-thiadiazol-2-yl)-3-phenylpyridin-2-yl]benzyl Base}amino)propan-1-one (3-5);

3-({4-[5-(5-amino-1,3,4-thiadiazol-2-yl)-3-phenylpyridin-2-yl]benzyl}amino)-1-phenylpropane -1-one;

or their stereoisomers.

In yet another embodiment, specific compounds of the invention include: 2-Chloro-5-phenyl-6-[4-({[4-(1,2,3-thiadiazol-4-yl)benzyl ]amino}methyl)phenyl]nicotinonitrile; (2-6) or a pharmaceutically acceptable salt or stereoisomer thereof.

In yet another embodiment, specific compounds of the invention include: 1-(2-aminophenyl)-3-({4-[5-(5-amino-1,3,4-thiadiazole-2- yl)-3-phenylpyridin-2-yl]benzyl}amino)propan-1-one (3-5); or a pharmaceutically acceptable salt or stereoisomer thereof.

The compounds of the present invention may have asymmetric centers, chiral axes and chiral planes (as described in: E.L. Eliel and S.H. Wilen, Stereochemistry of Carbon Conapounds, John Wiley & Sons, New York, 1994, pp. 1119-1190), may Racemates, racemic mixtures and all possible isomers of individual diastereoisomers and mixtures thereof, including optical isomers, exist and all such stereoisomers are included within the scope of the present invention .

In addition, the compounds disclosed herein may exist in the form of tautomers, and although only one tautomeric structure is described, both tautomeric forms are included within the scope of the present invention. For example, any claim to compound A below should be understood to include tautomeric structure B (and vice versa) and mixtures thereof. Both tautomeric forms of the benzimidazolone moiety are also included within the scope of the present invention.

Tetrazole exists as a mixture of 1H/2H tautomers. Tautomeric forms of the tetrazole moiety are also included within the scope of the invention.

When any variable (eg, ^R1 , ^R2 , ^Rz, etc.) occurs more than one time in any component, its definition on each occurrence is independent of its definition on every other occurrence. Also, combinations of substituents and variables are permissible only if such combinations result in stable compounds. Lines drawn from a substituent into a ring system indicate that the indicated bond may be attached to any substitutable ring atom. If the ring system is polycyclic, it means that the bond is only to any suitable carbon atom of the nearest ring.

It is understood that the substituents and substitution patterns on the compounds of the present invention can be selected by those of ordinary skill in the art to provide chemically stable compound. If a substituent is itself substituted by more than one group, it is understood that these groups can be on the same carbon atom or on different carbon atoms, so long as a stable structure is obtained. The phrase "optionally substituted with one or more substituents" is understood to be equivalent to the phrase "optionally substituted with at least one substituent", in which case an embodiment may have from zero to four substituents and another One embodiment may have from zero to three substituents.

"Alkyl" as used herein includes both branched and straight chain saturated aliphatic hydrocarbon groups containing the indicated number of carbon atoms. For example, C _{1 -C 10} in "C 1 -C ₁₀ alkyl" is defined as including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 group of carbon atoms. For example, "C ₁ -C ₁₀ alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl base, decyl, etc. The term "cycloalkyl" refers to a monocyclic saturated aliphatic hydrocarbon group containing the indicated number of carbon atoms. For example "cycloalkyl" includes cyclopropyl, methylcyclopropyl, 2,2-dimethylcyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl and the like.

"Alkoxy" means a cyclic or acyclic alkyl group containing the indicated number of carbon atoms attached through an oxygen bridge. Accordingly, "alkoxy" includes the above definitions for alkyl and cycloalkyl.

If no number of carbon atoms is specified, the term "alkenyl" refers to a straight chain, branched chain or cyclic non-aromatic hydrocarbon radical containing 2 to 10 carbon atoms and at least one carbon-carbon double bond. In one embodiment, there is one carbon-carbon double bond, and there may be up to four non-aromatic carbon-carbon double bonds. Thus, " _C2 - _C6 alkenyl" refers to an alkenyl group containing 2-6 carbon atoms. Alkenyl includes ethenyl, propenyl, butenyl, 2-methylbutenyl and cyclohexenyl. Straight chain, branched or cyclic moieties of alkenyl groups may contain double bonds, and if substituted alkenyl groups are indicated, such moieties may be substituted.

The term "alkynyl" refers to a straight chain, branched chain or cyclic hydrocarbon group containing 2 to 10 carbon atoms and at least one carbon-carbon triple bond. Up to three carbon-carbon triple bonds may be present. Thus, " _C2 - _C6alkynyl " refers to an alkynyl group containing 2-6 carbon atoms. Alkynyl includes ethynyl, propynyl, butynyl, 3-methylbutynyl, and the like. Straight chain, branched or cyclic moieties of an alkynyl group may contain a triple bond, and if indicated as a substituted alkynyl, such moieties may be substituted.

In some cases, substituents can be defined with a range of carbon atoms including zero, eg (C ₀ -C ₆ )alkenyl-aryl. If aryl is phenyl, then this definition would include _phenyl itself as well as _-CH2Ph , _-CH2CH2Ph , -CH( _CH3 ) _CH2CH ( _CH3 )Ph, etc.

"Aryl" as used herein means any stable monocyclic or bicyclic carbocyclic ring containing up to 7 atoms per ring, at least one of which is aromatic. Examples of such aryl moieties include phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl. Where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is through the aromatic ring.

The term "heterocycle" or "heterocyclyl" as used herein means a 3- to 10-membered aromatic or non-aromatic heterocyclic ring containing 1-4 heteroatoms selected from O, N and S, including bicyclic groups. Thus, "heterocyclyl" includes the aforementioned heteroaryl groups and their dihydro and tetrahydro analogs. Other examples of "heterocyclyl" include, but are not limited to, the following groups: benzimidazolyl, benzimidazolonyl, benzofuryl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, Benzothienyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furyl, imidazolyl, indolinyl, indolyl, indolazinyl (indolazinyl), indazolyl, Isobenzofuryl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl (naphthpyridinyl), oxadiazolyl, oxazolyl, oxazoline, isoxazoline, Oxetanyl, pyranyl, pyrazinyl, pyrazolopyrimidinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazoline Base, quinolinyl, quinoxalinyl, tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, 1 , 4-dioxanyl, hexahydroazepanyl, piperazinyl, piperidinyl, pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzimidazole Dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzoxazolyl, dihydrofuryl, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroiso Thiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolyl, Hydrogen tetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuryl and tetrahydrofuryl Hydrothienyl groups and their N-oxides. Attachment of a heterocyclyl substituent can occur through a carbon atom or through a heteroatom.

In another embodiment, the heterocyclyl group is selected from 2-azepine, benzimidazolyl, benzimidazolone, 2-diazapinone, imidazolyl, 2-imidazolidinone, indolyl, isoquinolinyl, morpholinyl, piperidinyl, piperazinyl, pyridyl, pyrrolidinyl, 2-piperidone, 2-pyrimidinone, 2-pyrrolidone, quinone Linyl, tetrazolyl, tetrahydrofuryl, tetrahydroisoquinolyl, thienyl, pyrazolopyrimidinyl, pyrazolyl, thiazolyl, oxadiazolyl and triazolyl.

Those of ordinary skill in the art will recognize that "halogen" as used herein includes chlorine, fluorine, bromine and iodine.

As used herein, substituted alkyl, substituted cycloalkyl, substituted aroyl, substituted aryl, substituted heteroaroyl, substituted heteroaryl, substituted arylsulfonyl, substituted heteroarylsulfonyl, and substituted heterocycle include compounds other than Moieties containing 1-4 substituents (in another embodiment 1-3 substituents) in addition to the point of attachment of the remaining moieties, unless specifically defined otherwise. Such substituents are selected from groups including, but not limited to, F, Cl, Br, CF ₃ , NH ₂ , N(C ₁ -C ₆ alkyl) ₂ , NO ₂ , CN, (C ₁ -C ₆ Alkyl) O-, (aryl) O-, -OH, O _a -P=O(OH) ₂ , (C ₁ -C ₆ alkyl) S(O) _m -, (C ₁ -C ₆ alkane Base) C(O)NH-, H ₂ NC(NH)-, (C ₁ -C ₆ alkyl)C(O)-, (C ₁ -C ₆ alkyl)OC(O)-, (C ₁ -C ₆ alkyl)OC(O)NH-, phenyl, pyridyl, imidazolyl, oxazolyl, isoxazolyl, tetrazolyl, thiazolyl, thienyl, furyl, isothiazolyl and C ₁ -C ₂₀ alkyl. For example, (C ₁ -C ₆ )alkyl may be selected from OH, oxo, halogen, alkoxy by one, two, three or four (in another embodiment by one, two or three) Substituents of radicals, dialkylamino groups or heterocyclic groups (such as morpholinyl, piperidinyl, etc.). In this case, if one substituent is oxo and the other is OH, the following groups fall within the above definition: -C=O) _CH2CH (OH) _CH3 , -(C=O)OH, _-CH2 (OH) _CH2CH (O), etc.

When R ³ and R ⁴ on the same carbon atom in the definition are combined to form -(CH ₂ ) _t -, the moiety formed is as follows:

In addition, such cyclic moieties may optionally include heteroatoms. Examples of such heteroatom-containing cyclic moieties include, but are not limited to:

In another embodiment, p is zero.

In another embodiment, q is zero.

In another embodiment, ^Ra is (C ₁ -C ₆ )alkyl.

In another embodiment, R ^b is independently selected from: H, (C ₁ -C ₆ )alkyl, (C═O)O(C ₁ -C ₆ )alkyl, (C═O)(C ₁ - C ₆ ) alkyl and S(O) ₂ R ^a .

In another embodiment, R ^z is selected from: C ₁ -C ₆ alkyl, -(C=O)(C ₁ -C ₆ )alkyl, halogen, phenyl, OH, O _a -P=O( OH) ₂ , NH ₂ and oxo.

In another embodiment, R ¹ is selected from: (C ₁ -C ₆ )alkyl, CO ₂ H, halogen, CN, OH, O _a -P=O(OH) ₂ , oxo, CHO and heterocycle base, the heterocyclic group is selected from:

is optionally substituted with one to three substituents selected from ^Rz .

In another embodiment, R ¹ is selected from: halogen, CN and

is optionally substituted with one to three substituents selected from ^Rz .

In another embodiment, R ² is selected from: (C ₁ -C ₆ )alkyl, CO ₂ H, halogen, CN, OH, O _a -P=O(OH) ₂ , oxo, CHO and heterocycle base.

In another embodiment, ^R3 and ^R4 are selected from: H and _-CH3 .

In another embodiment, ^R3 and ^R4 are selected from: H.

In another embodiment, Rz ^' is selected from: -(C=O)phenyl, phenyl, heterocyclyl and cycloalkyl optionally replaced by 1- 3 R ^z substitutions.

-(C＝O)苯基和环丙基，任选被一个至三个选自苯基、NH₂、卤素、C₁-C₆烷基和的取代基取代。In another embodiment, Rz ^' is selected from:

-(C=O)phenyl and cyclopropyl, optionally replaced by one to three selected from phenyl, NH ₂ , halogen, C ₁ -C ₆ alkyl and of substituents.

The present invention includes the free form of the compound of formula A as well as pharmaceutically acceptable salts and stereoisomers thereof. Certain isolated specific compounds exemplified herein are protonated salts of amine compounds. The term "free form" refers to the non-salt form of the amine compound. The pharmaceutically acceptable salts included herein include not only the isolated salts exemplified by the specific compounds described herein, but also all typical pharmaceutically acceptable salts of the free form of the compound of formula A. The free forms of specific salt compounds described herein can be isolated by techniques well known in the art. For example, the free form can be regenerated by treating the salt with a suitable dilute aqueous base, such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate. The free forms will differ to some extent in certain physical properties (such as solubility in polar solvents) relative to their respective salt forms, but the acid and base salts are pharmaceutically compatible with their respective salt forms for the purposes of the present invention. The free form of is comparable.

Pharmaceutically acceptable salts of the compounds of the present invention can be synthesized from compounds of the present invention which contain basic or acidic moieties by conventional chemical methods. In general, salts of basic compounds can be prepared by ion exchange chromatography or by reacting the free base with stoichiometric or excess amounts of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents. . Similarly, salts of acidic compounds may be formed by reaction with a suitable inorganic or organic base.

Accordingly, pharmaceutically acceptable salts of the compounds of the present invention include conventional non-toxic salts of the compounds of the present invention formed by reacting a basic compound of the present invention with an inorganic or organic acid. For example, conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc., and from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, Acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetyl Oxy-salts made from benzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, trifluoroacetic acid (TFA), etc.

When the compound of the present invention is an acidic compound, suitable "pharmaceutically acceptable salts" refer to salts prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganic, potassium, sodium, zinc, and the like. Particularly preferred are ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include those of primary, secondary, and tertiary amines, substituted amines (including naturally substituted amines), cyclic amines, and basic ion exchange resins, such as arginine, betaine, Caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N - Ethylpiperidine, Glucosamine, Glucosamine, Histidine, Hypamine, Isopropylamine, Lysine, Methylglucosamine, Morpholine, Piperazine, Piperidine, Polyamine Resin, Prole Caine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc.

The preparation of the aforementioned pharmaceutically acceptable salts and other typical pharmaceutically acceptable salts is more fully described by Berg et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977:66:1-19.

It should also be noted that the compounds of the present invention are potential internal salts or zwitterions, because under physiological conditions, the deprotonated acidic moiety (such as carboxyl group) in the compound can be anionic, and this charge can be protonated or alkylated. The cationic charge of the basic moiety (eg, quaternary ammonium nitrogen atom) balances out internally.

Uses

The compounds of the present invention are inhibitors of Akt activity and are therefore useful in the treatment of cancers, especially cancers associated with Akt activity disorders and Akt downstream cellular targets. These cancers include, but are not limited to, ovarian, pancreatic, breast, and prostate cancers, as well as cancers in which the tumor suppressor protein PTEN is mutated (including glioblastoma) (Cheng et al., Proc. Natl. Acad. Sci. (1992 ) 89: 9267-9271; Cheng et al., Proc. Natl. Acad. Sci. (1996) 93: 3636-3641; .Chem. (1999) 274: 21528-21532; Graff, Expert. Opin. Ther. Targets (2002) 6(1): 103-113; and Yamada and Araki, J. Cell Science (2001) 114: 2375-2382 ; Mischel and Cloughesy, Brain Pathol. (2003) 13(1):52-61).

It is particularly believed that the compounds, compositions and methods provided herein are useful in the treatment of cancers, including solid tumors, such as skin cancer, breast cancer, brain cancer, cervical cancer, testicular cancer, and the like. More specifically, cancers that may be treated with the compounds, compositions and methods of the present invention include, but are not limited to: Cardiac : Sarcomas (Angiosarcoma, Fibrosarcoma, Rhabdomyosarcoma, Liposarcoma), Myxoma, Rhabdomyosarcoma, Fibroma, Lipoma and teratomas; lung : bronchial carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar carcinoma (bronchiole carcinoma), bronchial adenocarcinoma, sarcoma, lymphoma, cartilage Hamartoma, mesothelioma; gastrointestinal : esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (gastric cancer, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, high Glucagonoma, gastrinoma, carcinoid tumor, VIP tumor), small bowel (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma ), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); genitourinary tract : kidney (adenocarcinoma, Wirm's tumor [Wilms tumor], lymphoma, leukemia) , bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, malignant teratoma, choriocarcinoma) , sarcoma, mesenchymal cell carcinoma, fibroid, fibroadenoma, adenomatoid tumor, lipoma); liver : hepatocellular carcinoma (liver cell carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma , hemangioma; bone : osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticular cell sarcoma), multiple myeloma, malignant giant cell Tumors chordoma, osteochondroma (exostoses of bone cartilage), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumor; nervous system : skull (osteoma, hemangioma, Granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningeal carcinoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, etympanoma, germ cell tumor [pineeal tumor], glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumor), spinal cord (neurofibroma, meningioma, glial tumor, sarcoma); gynecology : uterus (endometrial carcinoma), cervix (cervical carcinoma, preneoplastic cervical dysplasia), ovary (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified Carcinoma], granulosa cell-theca cell tumor, androcytoma, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tube carcinoma); hematology : blood (myelogenous leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia , myeloproliferative disorder, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; skin : malignant melanoma, basal cell carcinoma, squamous epithelium Cell carcinoma, Kaposi's sarcoma, dysplastic nevus, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and adrenal gland : neuroblastoma. Accordingly, the term "cancer cell" as provided herein includes cells affected by any of the aforementioned diseases.

Akt signaling regulates multiple key steps in the angiogenesis process. Shiojima and Walsh, Circ. Res. (2002) 90: 1243-1250. The utility of angiogenesis inhibitors in cancer therapy is known from the literature, see e.g. J. Rak et al., Cancer Research, 55:4575-4580, 1995 and Dredge et al., Expert Opin. Biol. Ther. (2002) 2 (8): 953-966. The role of angiogenesis in cancer has been shown in many types of cancers and tissues: breast cancer (G. Gasparini and A.L.Harris, J. Clin. Oncol., 1995, 13: 765-782; M. Toi et al., Japan .J.Cancer Res., 1994,85:1045-1049); Bladder cancer (A.J.Dickinson et al., Br.J.Urol., 1994,74:762-766); Colon cancer (L.M.Ellis et al., Surgery, 1996, 120(5):871-878); and Oral Tumors (J.K. Williams et al., Am. J. Surg., 1994, 168:373-380). Other cancers include advanced tumor, hairy cell leukemia, melanoma, advanced head and neck cancer, metastatic renal cell, non-Hodgkin's lymphoma, metastatic breast cancer, breast adenocarcinoma, advanced melanoma, pancreatic Carcinoma, gastric cancer, glioblastoma, lung cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, renal cell carcinoma, various solid tumors, multiple myeloma, metastatic prostate cancer, malignant glia tumor, renal carcinoma, lymphoma, refractory persistent disease, refractory multiple myeloma, cervical cancer, Kaposi's sarcoma, recurrent anaplastic glioma, and metastatic colon cancer (Dredge et al, Expert Opin. Biol. Ther. (2002) 2(8):953-966). Therefore, the Akt inhibitors disclosed in this application can also be used to treat these angiogenesis-related cancers.

Tumors that have undergone neovascularization exhibit increased metastatic potential. Indeed, angiogenesis is essential for tumor growth and metastasis. (S. P. Cunningham et al., Can. Research, 61:3206-3211 (2001)). Therefore, the Akt inhibitor disclosed in this application can also be used to prevent or reduce the metastasis of tumor cells.

Also included within the scope of this invention is a method of treating or preventing a disease involving angiogenesis comprising administering to a mammal in need of such treatment a therapeutically effective amount of a compound of this invention. An example of such a condition is ocular neovascular disease, in which much of the tissue damage is attributable to abnormal infiltration of ocular blood vessels (see WO 00/30651, published June 2, 2000). Adverse infiltration can be caused by ischemic retinopathy, such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, etc., or by degenerative diseases such as choroidal neovascularization seen in age-related macular degeneration. Therefore, inhibiting the growth of blood vessels by administering the compound of the present invention will prevent the infiltration of blood vessels, and prevent or treat diseases involving angiogenesis, such as retinal angiogenesis, diabetic retinopathy, age-related macular degeneration and other eye diseases.

Also included within the scope of this invention are methods of treating or preventing non-malignant diseases involving angiogenesis including, but not limited to: ocular diseases (e.g., retinal vascularization, diabetic retinopathy, age-related macular degeneration) , atherosclerosis, arthritis, psoriasis, obesity, and Alzheimer's disease (Dredge et al., Expert Opin. Biol. Ther. (2002) 2(8):953-966). In another embodiment, methods of treating or preventing diseases involving angiogenesis include ocular diseases (eg, retinal vascularization, diabetic retinopathy, age-related macular degeneration), atherosclerosis, arthritis, psoriasis.

Also included within the scope of the invention are methods of treating proliferative disorders such as restenosis, inflammation, autoimmune disease, and allergy/asthma.

Also included within the scope of this invention are methods of treating insulin hypersecretion.

In one embodiment of the invention, the compounds of the invention are selective inhibitors, the inhibitory potency of which is determined by the PH domain. In this embodiment, the compound has reduced or no in vitro inhibitory activity against a truncated Akt protein lacking the PH domain.

In yet another embodiment, the compound of the invention is selected from the group consisting of selective inhibitors of Akt1, selective inhibitors of Akt2, and selective inhibitors of both Akt1 and Akt2.

In another embodiment, the compound of the invention is selected from the group consisting of selective inhibitors of Akt1, selective inhibitors of Akt2, selective inhibitors of Akt3, and selective inhibitors of two of the three Akt isoforms.

In another embodiment, the compounds of the invention are selective inhibitors of all three isoforms of Akt, but not one, two or all of which have been modified to delete the PH domain, the hinge region, or both. and a selective inhibitor of Akt isoforms in the hinge region.

The invention also relates to a method of inhibiting Akt activity comprising administering a pharmaceutically effective amount of a compound of the invention to a mammal in need thereof.

The compounds of the present invention may be administered to mammals (including humans) alone or in combination with pharmaceutically acceptable carriers, excipients or diluents in pharmaceutical compositions according to standard pharmaceutical practice. The compounds can be administered orally, or by parenteral administration, including intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.

The pharmaceutical composition containing the active ingredient may be in a form suitable for oral administration, such as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules elixirs, syrups or elixirs. The composition for oral administration can be prepared according to any pharmaceutical composition preparation method known in the art, and this composition can contain one or more additives selected from sweeteners, flavoring agents, coloring agents and preservatives, to Pharmaceutically delicate and palatable preparations are provided. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents such as microcrystalline cellulose, croscarmellose sodium, corn starch or alginic acid; binders such as starch, gelatin, polyvinylpyrrolidone or acacia; and lubricants such as magnesium stearate, stearic acid or talc. Tablets may be uncoated, or may be coated by known techniques to mask the unpleasant taste of the drug, or to delay breakdown and absorption in the gastrointestinal tract, thereby providing sustained release over a prolonged period of time. For example, water soluble taste-masking materials such as hydroxypropylmethylcellulose or hydroxypropylcellulose, or time delay materials such as ethylcellulose, cellulose acetate-butyrate may be employed.

Formulations for oral administration may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with a water-soluble carrier such as polyethylene glycol. glycol) or an oily medium such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and acacia; dispersing or wetting agents can be are natural phospholipids, such as lecithin, or condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain fatty alcohols, such as heptadecenyloxycetate Heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols such as polyoxyethylene sorbitan monooleate, or ethylene oxide with partial esters derived from fatty acids and hexoses Condensation products of partial esters of alcohol anhydrides such as polyoxyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives, such as, for example, ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, one or more Sweeteners such as sucrose, saccharin, or aspartame.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or palm oil, or in a mineral oil such as liquid paraffin. Oily suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those described above and flavoring agents may be added to provide a palatable oral preparation. These compositions can be preserved by adding antioxidants such as butylated hydroxyanisole or alpha-tocopherol.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water comprise the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions can be preserved by the addition of antioxidants such as ascorbic acid.

The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as liquid paraffin, or a mixture of these oils. Suitable emulsifiers may be natural phospholipids, such as soybean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of said partial esters with ethylene oxide , such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavoring, preservative and antioxidant agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant.

The pharmaceutical compositions of the present invention may be in the form of sterile injectable aqueous solutions. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.

The sterile injectable preparation may also be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oily phase. For example, the active ingredient can first be dissolved in a mixture of soybean oil and lecithin. The oil solution is then introduced into a mixture of water and glycerin and processed to form a microemulsion.

Injectable solutions or microemulsions can be introduced into the patient's bloodstream by local bolus injection. Alternatively, it may be advantageous to administer solutions or microemulsions in such a way as to maintain a constant circulating concentration of the compounds of the invention. To maintain this constant concentration, a continuous intravenous delivery device can be used. An example of such a device is the Deltec CADD-PLUS ^™ Model 5400 Intravenous Pump.

The pharmaceutical compositions of the present invention may be in the form of sterile injectable aqueous or oily suspensions for intramuscular or subcutaneous administration. This suspension can be formulated according to the known technique using those suitable dispersing or wetting agents and suspending agents mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example a solution in 1,3-butanediol. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Compounds of formula A may also be presented in the form of suppositories for rectal administration. These compositions can be formulated by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such excipient materials include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.

For topical administration, creams, ointments, gels, solutions or suspensions containing the compound of formula A are used. (For purposes of this application, topical applications shall include mouthwashes and mouthwashes.)

The compounds of the present invention may be administered intranasally by topical use of suitable intranasal vehicles and delivery devices, or by transdermal routes using transdermal patches well known to those of ordinary skill in the art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen. The compounds of the present invention can also be administered in the form of suppositories using bases such as cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycols.

When administering the compositions of the present invention to human subjects, the daily dosage will usually be determined by the attending physician, who will generally vary the dosage used according to the age, weight and reactivity of the individual patient and the severity of the patient's symptoms.

In one embodiment, a suitable amount of an Akt inhibitor is administered to a mammal undergoing cancer treatment. The amount of inhibitor administered is from about 0.1 mg/kg body weight to about 60 mg/kg body weight per day, or from 0.5 mg/kg body weight to about 40 mg/kg body weight per day. Another treatment regimen comprising a composition of the invention comprises from about 0.01 mg to about 1000 mg of an Akt inhibitor. In another embodiment, the dose of Akt inhibitor used is from about 1 mg to about 1000 mg.

The compounds of the present invention may also be used in combination with known therapeutic and anticancer agents. For example, compounds of the present invention may be used in combination with known anticancer drugs. Combinations of the compounds disclosed herein with other anticancer or chemotherapeutic drugs are within the scope of the present invention. Examples of these agents are found in V.T. Devita and S. Hellman (eds.), Cancer Principles and Practice of Oncology, 6th Edition (15 February 2001), Lippincott Williams & Wilkins Publishers. One of ordinary skill in the art will be able to discern which combination of drugs would be useful based on the nature of the drugs and the cancer involved. Such anticancer agents include the following: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic/cytostatic agents, antiproliferative agents, prenyl-protein transfer Enzyme inhibitors, HMG-CoA reductase inhibitors and other angiogenesis inhibitors, inhibitors of cell proliferation and survival signaling, and drugs that interfere with cell cycle checkpoints. The compounds of the invention are particularly useful when co-administered with radiation therapy.

In one embodiment, the compounds of the present invention may also be used in combination with known anticancer drugs, including the following: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cell Toxic agents, antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors and other angiogenesis inhibitors.

"Estrogen receptor modulator" refers to a compound that interferes with or inhibits the binding of estrogen to the receptor, regardless of mechanism. Examples of estrogen receptor modulators include, but are not limited to, tamoxifen, raloxifene, edoxifene, LY353381, LY117081, toremifene, fulvestrant, 2,2-dimethylpropionic acid 4-[7-(2,2-Dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]- 2H-1-benzopyran-3-yl)-phenyl ester, 4,4'-dihydroxybenzophenone-2,4-dinitrophenylhydrazone and SH646.

"Androgen receptor modulator" refers to a compound that interferes with or inhibits the binding of androgen to the receptor, by whatever mechanism. Examples of androgen receptor modulators include finasteride and other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide, liarazole and abiraterone acetate.

"Retinoid receptor modulator" refers to a compound that interferes with or inhibits the binding of a retinoid to a receptor, regardless of mechanism. Examples of such retinoid receptor modulators include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid, α-difluoromethylornithine, ILX23-7553, trans -N-(4'-hydroxyphenyl) retinamide and N-4-carboxyphenyl retinamide.

"Cytotoxic agent/cytostatic agent" refers to a compound that causes cell death or inhibits cell proliferation mainly by directly interfering with the function of cells or inhibiting or interfering with cell meiosis, including alkylating agents, tumor necrosis factors, intercalating agents, hypoxic Activatable compounds, microtubule inhibitors/microtubule stabilizers, mitotic kinesin inhibitors, inhibitors of kinases involved in mitotic progression, inhibitors of kinases involved in growth factor and cytokine signaling pathways, antimetabolites, biological Response modifiers, hormonal/antihormonal therapeutics, hematopoietic growth factors, monoclonal antibody-targeted therapeutics, topoisomerase inhibitors, proteosome inhibitors, and ubiquitin ligase inhibitors.

Examples of cytotoxic/cytostatic agents include, but are not limited to, sertenef, cachectin, ifosfamide, tasonamine, lonidamine, carboplatin, hexamethylmelamine, prednimustine, Dibromodulcitol, ramustine, formustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfan tosylate, trofosfamide, nimustine , dibromospirochloramine, purantepa, lobaplatin, satraplatin, profiromycin (profiromycin), cisplatin, irovin, dexifosfamide, cis-amine dichloride (2-methyl -pyridine) platinum, benzylguanine, glufosfamide, GPX100, tetrachloride (trans, trans, trans)-bis-μ-(hexane-1,6-diamine)-μ-[diamine- Platinum(II)]bis[diamine(chloro)platinum(II)], diarizidinylspermine, arsenic trioxide, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethyl Xanthine, Zorubicin, Idarubicin, Daunorubicin, Bisantrene, Mitoxantrone, Pirarubicin, Pinafilide, Valrubicin, Amrubicin, Antineoplastic , 3′-deammonia-3′-morpholino-13-deoxo-10-hydroxycarmine, liposomal anthracycline (annamycin), grubicin, elinefade, MEN10755, 4-desmethoxy-3-deamino-3-aziridine-4-methylsulfonyl daunorubicin (see WO 00/50032), Raf kinase inhibitors (eg Bay 43-9006) and mTOR Inhibitors (such as Wyeth's CCI-779).

An example of a hypoxia activatable compound is tirapazamine.

Examples of proteosome inhibitors include, but are not limited to, lactacystin and MLN-341 (Velcade).

Examples of microtubule inhibitors/microtubule stabilizers include paclitaxel, vindesine sulfate, 3',4'-didehydro-4'-deoxy-8'-norvinblastine, docetaxel, rhizomycin , dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881, BMS184476, vinflunine, cryptophycin, 2,3,4,5,6-pentafluoro-N-(3- Fluoro-4-methoxyphenyl)benzenesulfonamide, anhydrovinblastine, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L - Prolyl-L-proline-tert-butyramide, TDX258, epothilone (see eg US Patents 6,284,781 and 6,288,237) and BMS188797. In one embodiment, epothilone is not included in the microtubule inhibitor/microtubule stabilizer.

Examples of topoisomerases are topotecan, hycaptamine, irinotecan, rubitecan, 6-ethoxypropionyl-3', 4'-O-exo-benzylidene-chartreusin ), 9-methoxy-N, N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H)propylamine, 1-amino-9-ethyl -5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]-indolizine[ 1,2b]quinoline-10,13(9H,15H)dione, letotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350, BNPI1100 , BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxan, 2′-dimethylamino-2′-deoxy-etoposide, GL331, N-[2-(dimethylamino)ethyl] -9-Hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide, asulacrine, (5a, 5aB, 8aa, 9b)-9-[2-[ N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydroxy-3,5-dimethoxyphenyl]-5,5a,6,8 , 8a, 9-hexahydrofuro(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxol-6-one, 2,3-( Methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium, 6,9-bis[(2-aminoethyl)amino]benzo[g ] Isoquinoline (isoguinoline)-5,10-dione, 5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazole And[4,5,1-de]acridin-6-one, N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthene- 4-ylmethyl]formamide, N-(2-(dimethylamino)ethyl)acridine-4-carboxamide, 6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy -7H-indeno[2,1-c]quinolin-7-one and demesna.

Examples of inhibitors of mitotic kinesins, especially human mitotic kinesins, are found in PCT publications WO 01/30768 and WO 01/98278 and in pending U.S. patent applications 60/338,779 (filed December 6, 2001), 60/ 338,344 (filed December 6, 2001), 60/338,383 (filed December 6, 2001), 60/338,380 (filed December 6, 2001), 60/338,379 (filed December 6, 2001) and 60/344,453 (filed November 7, 2001) as described. In one embodiment, inhibitors of mitotic kinesins include, but are not limited to, KSP inhibitors, MKLP1 inhibitors, CENP-E inhibitors, MCAK inhibitors, and Rab6-KIFL inhibitors.

"Inhibitors of kinases involved in mitotic progression" include, but are not limited to, inhibitors of aurora kinases, inhibitors of Polo-like kinases (PLK) (particularly PLK-1 inhibitors), bub-1 inhibitors, and bub-R1 inhibitors agent.

"Antiproliferative agents" include antisense RNA and DNA oligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001, and antimetabolites such as enoxitabine, carmofur, tegafur, pentostatin, Oxyfluridine, trimetrexate, fludarabine, capecitabine, galocitabine, cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed, paltitrexid, etimetrex, thiazofurin, Decitabine, noratrexed, pemetrexed, nelzarabine, 2'-deoxy-2'-methylene (methylidene) cytidine, 2'-fluoromethylene-2'-deoxycytidine, N -[5-(2,3-dihydro-benzofuryl)sulfonyl]-N'-(3,4-dichlorophenyl)urea, N6-[4-deoxy-4-[N2-[2 (E), 4(E)-tetradecenoyl]glycylamino]-L-glyceryl-B-L-mannosyl-heptopyranosyl]adenine, aplidine, ecteinascidin, koji Satabine, 4-[2-Amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-b][1,4]thiazin-6-yl- (S)-Ethyl]-2,5-thienyl-1-glutamic acid, aminopterin, 5-fluorouracil, aranosin, acetic acid 11-acetyl-8-(carbamoyloxymethyl)- 4-Formyl-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)-tetradec-2,4,6-trien-9-yl ester, Swainsonine Triol, Lometrexol, Dexrazoxane, Methioninase, 2′-Cyano-2′-Deoxy-N4-Palmitoyl-1-B-D-Arabinofuranosine, 3-Aminopyridine- 2-formaldehyde thiosemicarbazone and trastuzumab.

Examples of monoclonal antibody-directed therapeutics include therapeutics that link cytotoxic agents or radioisotopes to cancer cell-specific or target cell-specific monoclonal antibodies. Such examples include Bexxar.

"HMG-CoA reductase inhibitor" refers to an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase. Examples of HMG-CoA reductase inhibitors that may be used include, but are not limited to, lovastatin ( ^MEVACOR® ; see U.S. Patents 4,231,938, 4,294,926, and 4,319,039), simvastatin ( ^ZOCOR® ; see U.S. Patents 4,444,784, 4,820,850, and 4,916,239),普伐他汀(PRAVACHOL ^ ；参见美国专利4,346,227、4,537,859、4,410,629、5,030,447和5,180,589)、氟伐他汀(LESCOL ^ ；参见美国专利5,354,772、4,911,165、4,929,437、5,189,164、5,118,853、5,290,946和5,356,896)、阿托伐他汀( ^LIPITOR® ; see US Patents 5,273,995, 4,681,893, 5,489,691 and 5,342,952) and cerivastatin (also known as rivastatin and ^BAYCHOL® ; see US Patent 5,177,080). Structural formulas for these HMG-CoA reductase inhibitors and additional HMG-CoA reductase inhibitors that can be used in the methods of the invention are found in M. Yalpani "Cholesterol Lowering Drugs", Chemistry & Industry, pp. 85-89 (February 1996 5th) on page 87 and in US Patents 4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitors as used herein includes all pharmaceutically acceptable lactones and ring-opened acid forms (i.e. the lactone ring opens to form the free acid) as well as salt and ester forms of compounds having HMG-CoA reductase inhibitory activity , therefore, the use of these salts, esters, ring-opening acids and lactone forms is included within the scope of the present invention.

"Prenyl protein transferase inhibitor" refers to a compound that inhibits any one or any combination of prenyl protein transferases, including farnesyl protein transferase (FPTase), I Type geranylgeranyl protein transferase (GGPTase-I) and type II geranylgeranyl protein transferase (GGPTase-II, also known as Rab GGPTase).

Examples of prenyl protein transferase inhibitors are found in the following publications and patents: WO 96/30343, WO 97/18813, WO 97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98 /29119, WO 95/32987, U.S. Patent 5,420,245, U.S. Patent 5,523,430, U.S. Patent 5,532,359, U.S. Patent 5,510,510, U.S. Patent 5,589,485, U.S. Patent 5,602,098, European Patent Publication 0 618 221, European Patent Publication 0 675 112, European Patent Publication 0 604 181, European Patent Publication 0 696 593, WO 94/19357, WO 95/08542, WO 95/11917, WO 95/12612, WO 95/12572, WO 95/10514, US Patent 5,661,152, WO 95/ 10515, WO 95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO 96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456 , WO 96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO 96/00736, U.S. Patent 5,571,792, WO 96/17861, WO 96/33159, WO 96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO 96/30362, WO 96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO 96/31501, WO 97/00252, WO 97 /03047, WO 97/03050, WO 97/04785, WO 97/02920, WO 97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO 98/02436 and U.S. Patent 5,532,359 . For an example of the effect of prenyl protein transferase inhibitors on angiogenesis, see European J. of Cancer, Vol. 35, No. 9, pp. 1394-1401 (1999).

"Angiogenesis inhibitor" refers to a compound that inhibits the formation of new blood vessels, regardless of mechanism. Examples of angiogenesis inhibitors include, but are not limited to, tyrosine kinase inhibitors such as tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1/KDR (VEGFR2) inhibitors, epidermis-derived, fibroblast-derived or platelet-derived growth factor inhibitors, MMP (matrix metalloproteinase) inhibitors, integrin blockers, interferon-alpha, interleukin-12, pentosan polysulfate, cyclooxygenase inhibitors , including nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen and selective cyclooxygenase-2 inhibitors such as celecoxib and rofecoxib (PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69, p. 475 (1982); Arch.Opthalmol., Vol. 108, p. 573 (1990); Anat. Rec., Vol. 238, p. 68 (1994); FEBS Letters, p. 372 Vol. 83 (1995); Clin, Orthop. Vol. 313, p. 76 (1995); J. Mol. Endocrinol., Vol. 16, p. 107 (1996); Jpn.J.Pharmacol., pp. Vol. 75, p. 105 (1997); Cancer Res., vol. 57, p. 1625 (1997); Cell, vol. 93, p. 705 (1998); Intl.J.Mol.Met., vol. 2 , p. 715 (1998); J.Biol.Chem., vol. 274, p. 9116 (1999)), steroid anti-inflammatory drugs (such as corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone pine dragon, methylprednisolone, betamethasone), carboxyamide triazole, combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol (fumagillol), thalidomide, Angiostatin, troponin-1, angiotensin II antagonists (see Femandez et al., J.Lab.Clin.Med. 105:141-145 (1985)) and anti-VEGF antibodies (see Nature Biotechnology, Vol. 17 , pp. 963-968 (October 1999); Kim et al., Nature, 362, 841-844 (1993); WO 00/44777; and WO 00/61186).

Other therapeutic agents capable of modulating or inhibiting angiogenesis and which may also be used in combination with the compounds of the present invention include those capable of modulating or inhibiting the blood coagulation and fibrinolytic systems (see Clin. Chem. La. Med. 38:679-692( 2000) for a review). Examples of such drugs that modulate or inhibit the coagulation and fibrinolytic pathways include, but are not limited to, heparins (see Thromb. Haemost 80:10-23 (1998)), low molecular weight heparins, and carboxypeptidase U inhibitors (also known as Active thrombin activates an inhibitor of fibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354 (2001)). Inhibitors of TAFIa have been described in US Patent Nos. 60/310,927 (filed August 8, 2001) and 60/349,925 (filed January 18, 2002).

A "drug that interferes with a cell cycle checkpoint" refers to a compound that inhibits protein kinases that transduce cell cycle checkpoint signals, thereby sensitizing cancer cells to DNA damaging agents. Such drugs include ATR inhibitors, ATM inhibitors, Chk1 and Chk2 kinase inhibitors, and cdk and cdc kinase inhibitors. Specific examples include 7-hydroxystaurosporine, flavopiridol (flavonoid antineoplastic drugs), CYC202 (Cyclacel) and BMS-387032.

"Inhibitors of cell proliferation and survival signaling pathways"refers to compounds that inhibit signal transduction cascades downstream of cell surface receptors. Such drugs include serine/threonine kinase inhibitors (including but not limited to Akt inhibitors as described in WO 02/083064, WO 02/083139, WO 02/083140 and WO 02/083138), Raf kinase inhibitors (such as BAY-43-9006), MEK inhibitors (such as CI-1040 and PD-098059), mTOR inhibitors (such as Wyeth CCI-779), and PI3K inhibitors (such as LY294002).

As mentioned above, combinations with NSAIDs involve the use of NSAIDs as potent COX-2 inhibitors. For the purposes of this specification, NSAIDs are effective with an _IC50 for COX-2 inhibition of 1 [mu]M or less as determined by cellular or microsomal assays.

The invention also includes combinations with NSAIDs which are selective COX-2 inhibitors. For the purposes of this specification, NSAIDs that are COX-2 selective inhibitors are defined as evaluated by cellular or microsomal assays by determining the ratio of their _IC50 for COX-2 to their _IC50 for COX-1, and their effect on COX-1. NSAIDs whose inhibition specificity for -2 is at least 100-fold higher than for COX-1. Such compounds include, but are not limited to, those described in U.S. Patent 5,474,995, U.S. Patent 5,861,419, U.S. Patent 6,001,843, U.S. Patent 6,020,343, U.S. Patent 5,409,944, U.S. Patent 5,436,265, U.S. Patent 5,536,752, U.S. Patent 5,550,142, U.S. Patent 5,604,260, U.S. Patent 5,698,584, 5,710,140, WO 94/15932, US Patent 5,344,991, US Patent 5,134,142, US Patent 5,380,738, US Patent 5,393,790, US Patent 5,466,823, US Patent 5,633,272 and US Patent 5,932,598, which are incorporated herein by reference.

Particularly useful COX-2 inhibitors in the methods of treatment of the present invention are: 3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone; and 5-chloro- 3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridyl)pyridine; or their pharmaceutically acceptable salts.

Compounds which have been described as specific COX-2 inhibitors and thus are useful in the present invention include, but are not limited to, the following: parecoxib, BEXTRA ^(R) and CELEBREX ^(R) , or pharmaceutically acceptable salts thereof.

Other examples of angiogenesis inhibitors include, but are not limited to, endostatin, ukrain, leopard enzyme, IM862, (chloroacetyl)carbamate 5-methoxy-4-[2-methyl-3-(3-methyl -2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl ester, acetyldinanaline, 5-amino-1-[[3,5-dichloro-4- (4-Chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide, CM101, squalamine, combretastatin, RPI4610, NX31838, mannopentose sulfate Phosphate, 7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]bis-(1 , 3-naphthalene disulfonate) and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone (SU5416).

"Integrin blocker" as used above refers to a compound that selectively antagonizes, inhibits or blocks the binding of a physiological ligand to α _v β ₃ integrin, selectively antagonizes, inhibits or blocks the binding of a physiological ligand to α _v β ₅ Integrin-binding compounds, compounds that antagonize, inhibit or block the binding of physiological ligands to both α _v β ₅ integrin and α _v β ₅ integrin, also refers to antagonize, inhibit or block the expression on capillary endothelial cells Compounds with specific integrin activity. _The term _also refers to antagonists _{of αvβ6} , _{αvβ8 , α1β1} , _{α2β1 , α5β1 , α6β1} and _α6β4 integrins _. The term also refers to α _v β ₃ , α _v β ₅ , α _v β ₆ , α _v β ₈ , α ₁ β ₁ , α ₂ β ₁ , α ₅ β ₁ , α ₆ β ₁ and α ₆ β ₄ integrants Antagonists of any combination of proteins.

Specific examples of tyrosine kinase inhibitors include N-(trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide, 3-[(2,4-dimethylpyrrol-5-yl )methylindenyl]indolin-2-one, 17-(allylamino)-17-demethoxygeldanamycin, 4-(3-chloro-4-fluorophenylamino) -7-methoxy-6-[3-(4-morpholinyl)propoxy]quinazoline, N-(3-ethynylphenyl)-6,7-bis(2-methoxyethyl Oxy)-4-quinazolinamine, BIBX1382, 2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-ring Oxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazepine -1-one, SH268, genistein, STI571, CEP2563, 4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d] methanesulfonate Pyrimidine, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, 4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline Azoline, SU6668, STI571A, N4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine and EMD 121974.

Combinations with compounds other than anticancer compounds are also within the scope of the methods of the invention. For example, combinations of compounds claimed herein with PPAR-gamma (ie, PPAR-gamma) agonists and PPAR-delta (ie, PPAR-delta) agonists are useful in the treatment of certain malignancies. PPAR-γ and PPAR-δ are nuclear peroxisome proliferator-activated receptors γ and δ. The expression of PPAR-γ on endothelial cells and its involvement in angiogenesis have been reported in the literature (see J. Cardiovasc. Pharmacol. 1998; 31: 909-913; J. Biol. Chem. 1999; 274: 9116 -9121; Invest. Ophthalmol Vis. Sci. 2000; 41:2309-2317). PPAR-gamma agonists were recently revealed to inhibit angiogenesis in response to VEGF in vitro; both troglitazone and rosiglitazone maleate inhibited the development of retinal neovascularization in mice. (Arch. Ophthamol. 2001; 119:709-717). Examples of PPAR-γ agonists and PPAR-γ/α agonists include, but are not limited to, thiazolidinediones (such as DRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone), fenofibrate , gemfibrozil, clofibrate, GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552-[(926, 5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropanoic acid (disclosed in USSN 09/782,856) and 2 (R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchromane (chromane)-2-carboxylic acid (Disclosed in USSN 60/235,708 and 60/244,697).

Another embodiment of the present invention is the use of the compounds disclosed herein in combination with gene therapy in the treatment of cancer. For reviews of genetic strategies for cancer treatment see Hall et al. (Am.J.Hum.Genet. 61:785-789, 1997) and Kufe et al. (Cancer Medicine, 5th ed., pp. 876-889, BC Decker, Hamilton 2000). Gene therapy can be used to deliver any tumor suppressor gene. Examples of such genes include, but are not limited to, p53 that can be delivered by recombinant virus-mediated gene transfer (see, e.g., U.S. Patent 6,069,134), uPA/uPAR antagonists (“Adenovirus-Mediated Delivery of a uPA/uPARAntagonist Suppresses Angiogenesis-Dependent Tumor Growth and Dissemination in Mice," Gene Therapy, 1998 Aug; 5(8): 1105-13) and gamma interferon (J. Immunol. 2000; 164: 217-222).

The compounds of the present invention may also be administered in combination with inhibitors of intrinsic multidrug resistance (MDR), especially MDR associated with high expression levels of transporters. Such MDR inhibitors include inhibitors of p-glycoprotein (P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853, and PSC833 (valsporda).

The compounds of this invention may be used in combination with antiemetics for the treatment of nausea and vomiting, including acute, delayed, late and pre-emesis, which may be caused by the compounds of this invention alone or in combination with radiation therapy . For the prevention or treatment of emesis, the compounds of the present invention can be used in combination with other antiemetics, especially neurokinin-1 receptor antagonists, 5HT3 receptor antagonists such as ondansetron, granisetron, tropisetron and zatisetron, GABAB receptor agonists such as baclofen, corticosteroids such as Decadron (dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten or, for example, in U.S. Patents 2,789,118, 2,990,401, 3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928, Other corticosteroids, antidopaminergic agents such as phenothiazines (eg, prochlorperazine, fluphenazine, thioridazine, and mesoridazine), metoclopramide, or dronabinol disclosed in 3,749,712. In another embodiment, combination therapy with antiemetics selected from the group consisting of neurokinin-1 receptor antagonists, 5HT3 receptor antagonists and corticosteroids is disclosed for the treatment or prophylaxis of resulting in vomiting.

Neurokinin-1 receptor antagonists that can be used in combination with the compounds of the present invention are fully described in, for example, U.S. Patents 5,162,339; 5,232,929; 5,242,930; 5,373,003; Patent publications EP 0 360 390, 0 394 989, 0 428 434, 0 429366, 0 430 771, 0 436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0 512 902 . 545 478, 0 558 156, 0 577 394, 0 585 913, 0 590 152, 0 599 538, 0 610 793, 0 634 402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 04699 67 , 0 707 006, 0 708 101, 0 709 375, 0 709 376, 0 714 891, 0 723 959, 0 733 632 and 0 776 893; PCT International Patent Publications WO 90/05525, 90/05729, 91/09844 , 91/18899, 92/01688, 92/06079, 92/12151, 92/15585, 92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330, 93/00331, 93 /01159, 93/01165, 93/01169, 93/01170, 93/06099, 93/09116, 93/10073, 93/14084, 93/14113, 93/18023, 93/19064, 93/21155, 93/21181 , 93/23380, 93/24465, 94/00440, 94/01402, 94/02461, 94/02595, 94/03429, 94/03445, 94/04494, 94/04496, 94/05625, 94/07843, 94 /08997, 94/10165, 94/10167, 94/10168, 94/10170, 94/11368, 94/13639, 94/13663, 94/14767, 94/15903, 94/19320, 94/19323, 94/20500 , 94/26735, 94/26740, 94/29309, 95/02595, 95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549, 95/11880, 95/14017, 95 /15311, 95/16679, 95/17382, 95/18124, 95/18129, 95/19344, 95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418, 95/30674 , 95/30687, 95/33744, 96/05181, 96/05193, 96/05203, 96/06094, 96/07649, 96/10562, 96/16939, 96/18643, 96/20197, 96/21661, 96 /29304, 96/29317, 96/29326, 96/29328, 96/31214, 96/32385, 96/37489, 97/01553, 97/01554, 97/03066, 97/08144, 97/14671, 97/17362 , 97/18206, 97/19084, 97/19942 and 97/21702; and British Patent Publications 2 266 529, 2 268 931, 2 269 170, 2 269 590, 2 271774, 2 292 144, 2 293 168, 2 293 169 and 2 302 689. Methods for the preparation of these compounds are fully described in the aforementioned patents and publications, which are incorporated herein by reference.

In another embodiment, the neurokinin-1 receptor antagonist used in combination with the compounds of the invention is selected from the group consisting of: 2-(R)-(1-(R)-(3,5-bis(trifluoromethyl ) phenyl) ethoxy) -3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-1H, 4H-1,2,4-triazolo) methyl ) morpholine or a pharmaceutically acceptable salt thereof, which is described in US Patent No. 5,719,147.

The compounds of the invention may also be administered with drugs useful in the treatment of anemia. Such drugs for treating anemia are, for example, continuous erythropoiesis receptor activators (such as epoetin alfa).

The compounds of the present invention may also be administered with drugs useful in the treatment of neutropenia. Such neutropenia therapeutic agents are, for example, hematopoietic growth factors that regulate neutrophil production and function, such as human granulocyte colony-stimulating factor (G-CSF). Examples of G-CSF include filgrastim.

The compounds of the present invention can also be administered together with immune enhancing drugs such as levamisole, isoprinosine and Zidaxian.

Accordingly, the scope of the present invention includes the use of the claimed compounds in combination with a second compound selected from: 1) estrogen receptor modulators, 2) androgen receptor modulators, 3) retinoids receptor modulators, 4) cytotoxic/cytostatic agents, 5) antiproliferative agents, 6) prenyl protein transferase inhibitors, 7) HMG-CoA reductase inhibitors, 8) HIV protease inhibitors, 9) reverse transcriptase inhibitors, 10) angiogenesis inhibitors, 11) PPAR-γ agonists, 12) PPAR-δ agonists, 13) intrinsic multidrug resistance inhibitors, 14) antiemetics, 15) for Drugs to treat anemia, 16) drugs to treat neutropenia, 17) immune enhancing drugs, 18) inhibitors of cell proliferation and survival signaling, 19) drugs that interfere with cell cycle checkpoints.

The term "administration" or "administering" and grammatical variants thereof (eg "administering" a compound) in reference to a compound of the invention means introducing the compound or a prodrug of the compound into the system of an animal in need of treatment. When a compound of the present invention or a prodrug thereof is provided together with one or more other active agents (e.g., cytotoxic agents, etc.), "administration" or "administering" and grammatical variants thereof are each understood to include simultaneous and sequential introduction Compounds or their prodrugs and other drugs.

As used herein, the term "composition" includes a product that contains the specified ingredients in the specified amounts, as well as any product that combines, directly or indirectly, the specified ingredients in the specified amounts.

The term "therapeutically effective amount" as used herein refers to the amount of an active compound or agent that can cause a biological or medical response in a tissue, system, animal or human body being investigated by a researcher, veterinarian, hospital doctor or other clinician.

The term "treating cancer" or "cancer treatment" refers to administration to a mammal suffering from cancer, and also refers to the effect of alleviating the condition of the cancer by killing cancer cells, and also refers to the effect of causing the growth and/or metastasis of the cancer to be inhibited.

In one embodiment, the angiogenesis inhibitor used as the second compound is selected from the group consisting of tyrosine kinase inhibitors, inhibitors of epidermis-derived growth factor, inhibitors of fibroblast-derived growth factor, platelet-derived growth factor Factor inhibitors, MMP (matrix metalloproteinase) inhibitors, integrin blockers, alpha interferon, interleukin-12, pentosan polysulfate, cyclooxygenase inhibitors, carboxyamide triazole, Combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin, troponin-1, or anti-VEGF antibody. In one embodiment, the estrogen receptor modulator is tamoxifen or raloxifene.

Also included within the scope of the claimed invention is a method of treating cancer comprising administering a therapeutically effective amount of a compound of formula A in combination with radiation therapy and/or in combination with a second compound selected from the group consisting of: 1) estrogen Hormone receptor modulators, 2) androgen receptor modulators, 3) retinoid receptor modulators, 4) cytotoxic/cytostatic agents, 5) antiproliferative agents, 6) prenyl protein transfer Enzyme inhibitors, 7) HMG-CoA reductase inhibitors, 8) HIV protease inhibitors, 9) reverse transcriptase inhibitors, 10) angiogenesis inhibitors, 11) PPAR-γ agonists, 12) PPAR-δ agonists 13) Inherent multidrug resistance inhibitors, 14) Antiemetics, 15) Drugs used to treat anemia, 16) Drugs used to treat neutropenia, 17) Immunoenhancing drugs, 18) Cell proliferation and inhibitors of survival signaling, 19) drugs that interfere with cell cycle checkpoints.

Yet another embodiment of the present invention is a method of treating cancer comprising administering a therapeutically effective amount of a compound of formula A in combination with paclitaxel or trastuzumab.

The present invention also includes a method of treating or preventing cancer comprising administering a therapeutically effective amount of a compound of formula A in combination with a COX-2 inhibitor.

The present invention also includes a pharmaceutical composition for treating or preventing cancer, said composition comprising a therapeutically effective amount of a compound of formula A and a second compound selected from the group consisting of: 1) an estrogen receptor modulator, 2) an androgen Receptor Modulators, 3) Retinoid Receptor Modulators, 4) Cytotoxic/Cytostatic Agents, 5) Antiproliferative Agents, 6) Prenyl Protein Transferase Inhibitors, 7) HMG-CoA Reduction Enzyme inhibitors, 8) HIV protease inhibitors, 9) reverse transcriptase inhibitors, 10) angiogenesis inhibitors, 11) PPAR-γ agonists, 12) PPAR-δ agonists, 13) cell proliferation and survival signaling 14) Drugs that interfere with cell cycle checkpoints.

All patents, publications and pending patent applications identified herein are hereby incorporated by reference.

The abbreviations used in the chemical expressions and in the following examples are: AEBSF (p-aminoethylbenzenesulfonyl fluoride); BSA (bovine serum albumin); CuI (cuprous iodide); CuSO ₄ (copper sulfate); DCE ( Dichloroethane); DCM (dichloromethane); DIEPA (diisopropylacetamide); DMF (dimethylformamide); DMSO (dimethylsulfoxide); DTT (dithiothreitol); EDTA (ethylenediaminetetraacetic acid); EGTA (ethylene glycol tetraacetic acid); EtOAc (ethyl acetate); EtOH (ethanol); HOAc (acetic acid); HPLC (high performance liquid chromatography); HRMS (high resolution mass spectrometry); LCMS (liquid chromatography-mass spectrometry); MeOH (methanol); MP (methyl polystyrene); NaHCO ₃ (sodium bicarbonate); Na ₂ SO ₄ (sodium sulfate); NH ₄ OAc (ammonium acetate); NBS (N-bromosuccinimide); NMR (nuclear magnetic resonance); PBS (phosphate-buffered saline); PCR (polymerase chain reaction); PS-DIEA (polystyrene diisopropylethylamine); THF ( tetrahydrofuran); TFA (trifluoroacetic acid).

Compounds of the present invention can be prepared by using the reactions shown in the following reaction schemes, in addition to other standard procedures known in the literature or exemplified in the test methods. Accordingly, the illustrative reaction schemes that follow are not limited by the compounds listed or by any particular substituents used for illustration purposes. The substituent numbering shown in the reaction schemes does not necessarily correspond to the numbering used in the claims, and where multiple substituents are permitted as defined in Formula A above, the individual substituents attached to the compound are generally indicated for clarity. base.

The reaction used to produce the compound of the present invention can be carried out by other standard operating methods known in the literature or exemplified in the test methods such as ester hydrolysis, cleavage of protecting groups, etc. reaction to proceed.

Brief description of the reaction process

Reaction Scheme I illustrates the preparation of compounds of the invention starting from appropriately substituted hydroxypyridines I-1. This starting material can be converted to the corresponding pyridinium triflate 1-2 by treatment with triflic anhydride in pyridine. Intermediate 1-2 can be subjected to standard Suzuki coupling reactions with functionalized arylboronic acids to yield 1-3. Parallel reductive amination of 1-3 with various amines (HNR ⁶ R ⁷ , as exemplified by compounds in Scheme 1 and Table 1) and polymer-supported borohydrides then afforded 1-4.

Reaction Scheme II illustrates the synthesis of compounds of the present invention, from ketones II- 1 start. Condensation of II-1 with N,N-dimethylformamide dimethyl acetal gives keto-enamine II-2, which cyclizes with 2-cyanoacetamide to give pyridone II-3. Treatment of II-3 with phosphorous oxychloride yields chloropyridine II-4. The latter undergoes radical bromination followed by displacement with an appropriately substituted amine to yield amine II-5.

Reaction Scheme III illustrates the displacement of chlorine with various amines (NR'R") and alkoxides (OR'), which are well known in the chemical arts.

Reaction Scheme IV illustrates the synthesis of compounds of the present invention from ketones IV- 1 start. Reaction of IV-1 with 2-chloro-N,N-dimethylaminoallylidenedimethylamine hexafluorophosphate yields IV-2, which is chemically converted to cyanopyridine IV-3 by palladium. Free radical bromination of IV-3 followed by displacement with an appropriately substituted amine yields amine IV-4. The cyano group of IV-4 is then converted to a heterocycle and amide using reactions well known in the chemical arts.

Reaction scheme I

Reaction scheme II

Reaction scheme III

Reaction scheme IV

Example

The following examples are provided to assist in further understanding of the present invention. The particular materials, forms and conditions employed are intended to further illustrate the invention and not to limit the reasonable scope of the invention. The reagents used in the synthesis of the compounds shown in the table below are either commercially available or readily prepared by one of ordinary skill in the art.

Process 1

5-cyano-3-phenylpyridin-2-yl trifluoromethanesulfonate (1-2)

At 0°C, trifluoromethanesulfonic anhydride (3.384 g, 1.20 mmol) was added dropwise with stirring to 6-hydroxy-5-phenylnicotinonitrile (1-1) (1.962 g, 10.0 mmol, 60 mL of anhydrous pyridine solution) . After addition of trifluoromethanesulfonic anhydride, the pyridine solution was stirred overnight as the temperature rose from 0°C to room temperature. The next morning, the pyridine solution was dissolved in DCM (300 mL), washed with water, saturated NaHCO ₃ and brine, and dried over anhydrous Na ₂ SO ₄ . Filtration and concentration gave crude product which was purified by flash silica gel chromatography to give pure 5-cyano-3-phenylpyridin-2-yl triflate (1-2). Analytical LCMS: singlet (214nm), 3.553 min. ¹ H NMR (500MHz, CDCl ₃ ): δ8.64(d, J=2.2Hz, 1H), 8.16(d, J=2.2Hz, 1H), 7.52-7.56(m, 3H), 7.45-7.49(m , 2H).

6-(4-Formylphenyl)-5-phenylnicotinonitrile (1-3)

5-cyano-3-phenylpyridin-2-yl trifluoromethanesulfonate (1-2; 328 mg, 1.0 mmol), 4-formylphenyl A mixture of boronic acid (225 mg, 1.5 mmol), Pd( _dppf )( _CH2Cl2 ) (16 mg, 0.02 mmol), _2M aqueous _Cs2CO3 (2 mL, 4 mmol) and THF (2 mL) for 10 min. After the reaction mixture was cooled to room temperature, it was treated with DCM (40 mL), then washed with water, brine, and dried over _{anhydrous Na2SO4} . Filtration and concentration gave crude product which was purified by flash silica gel chromatography to give pure 6-(4-formylphenyl)-5-phenylnicotinonitrile (1-3). Analytical LCMS: singlet (214nm), 3.240 min. ¹ H NMR (500MHz, CDCl ₃ ): δ10.01(s, 1H), 8.92(d, J=2.0Hz, 1H), 8.04(d, J=2.0Hz, 1H), 7.79(d, J=8.2 Hz, 2H), 7.55 (d, J=8.2Hz, 1H), 7.31-7.39 (m, 3H), 7.13-7.18 (m, 2H).

5-Phenyl-6-[4-({[4-(1,2,3-thiadiazol-4-yl)benzyl]amino}methyl)phenyl]nicotinonitrile (1-5)

At room temperature, 6-(4-formylphenyl)-5-phenylnicotinonitrile (1-3; 57 mg, 0.2 mmol), 1-[4-(1,2,3-thiadiazole-4 -yl)phenyl]methylamine (1-4; 77mg, 0.4mmol) and MP-BH ₃ (CN) resin (1g, 1.2mmol) in DCE-HC(OMe) ₃ -HOAc-(7:2:1 , 6 mL) was shaken overnight. When complete, the resin was filtered, washing with MeOH (2 x 4 mL). The combined solution was concentrated and purified by LCMS to give the desired pure product (TFA salt) as a pale yellow solid (1-5). Analytical LCMS: singlet (214nm), 2.538 min. ¹ H NMR (500MHz, DMSO-d ₆ ): δ10.94(s, 1H), 9.69(s, 1H), 9.13(d, J=2.0Hz, 1H), 8.42(d, J=2.0Hz, 1H ), 8.22(d, J=8.3Hz, 2H), 7.65(d, J=8.3Hz, 2H), 7.39-7.45(m, 4H), 7.34-7.37(m, 3H), 7.25-7.27(m, 2H), _4.26 (s, 1H), 4.22(s, 1H); HRMS, _calcd for _C28H21N5S (M+1) 460.1591; found 460.1580.

1-{4-[3-phenyl-5-(1H-tetrazol-5-yl)pyridin-2-yl]phenyl}-N-[4-(1,2,3-thiadiazole-4 - Base) benzyl] methylamine (1-6)

5-Phenyl-6-[4-({[4-(1,2,3-thiadiazol-4-yl)benzyl]amino}methyl was treated with microwave (Smith-Synthesizer) at 165°C ) A mixture of phenyl]nicotinonitrile (1-5; TFA salt) (69 mg, 0.1 mmol), 2M aqueous NaN ₃ (0.75 mL, 1.5 mmol), 2M aqueous ZnBr ₂ (0.5 mL, 1.0 mmol) for 15 min. The solution was then concentrated. The residue was redissolved in DMSO (1 mL) and purified by LCMS to give the desired pure product (TFA salt) as a pale yellow solid (1-6). Analytical LCMS: singlet (214nm), 2.339 min. ¹ H NMR (500MHz, DMSO-d ₆ ): δ9.70(s, 1H), 9.32(d, J=2.1Hz, 1H), 8.42(d, J=2.1Hz, 1H), 8.22(d, J =8.2Hz, 2H), 7.68(d, J=8.3Hz, 2H), 7.42-7.48(M, 4H), 7.35-7.40(m, 3H), 7.28-7.32(m, 2H), 4.26(s, 2H), 4.23 (s, 2H); _{HRMS ,} calcd. for _C28H22N8S (M+1) 503.1761; found 503.1769.

Process 2

3-(Dimethylamino)-1-(4-methylphenyl)-2-phenylprop-2-en-1-one (2-2)

At 110°C, 1-(4-methylphenyl)-2-phenylethanone (2-1) (Renault, O; Dallemagne, P.; and Rault, S.Org.Prep.Proced.Int .1999, 31, 324) (3.29 g, 15.65 mmol) and N,N-Dimethylformamide dimethyl acetal (4.66 g, 39.12 mmol) in DMF (15 mL) was stirred for 1 hour. The resulting mixture was concentrated in vacuo to afford 3-(dimethylamino)-1-(4-methylphenyl)-2-phenylprop-2-en-1-one. LRMS m/z (M+H) calculated: 266.4, found: 266.2.

6-(4-methylphenyl)-2-oxo-5-phenyl-1,2-dihydropyridine-3-carbonitrile (2-3)

To a slurry of NaH (1.38 g in mineral oil, 60%, 34.41 mmol) in DMF (20 mL) was added 3-(dimethylamino)-1-(4-methylphenyl) dropwise over 40 min at 0 °C - 2-Phenylprop-2-en-1-one (2-2; 4.15 g, 15.64 mmol) and 2-cyanoacetamide (1.45 g, 17.2 mmol) in MeOH (1.5 mL) and DMF (40 mL) solution. The resulting mixture was stirred at 90°C for 4 hours, then cooled to room temperature. The mixture was poured into dilute aqueous HCl (240 mL, 0.25 M), filtered, washed with water (40 mL), and dried in vacuo to give 6-(4-methylphenyl)-2-oxo-5-phenyl-1, 2-Dihydropyridine-3-carbonitrile (2-3). ¹ H NMR (500 MHz, CDCl ₃ ): δ7.96 (s, 1H), 7.15-7.28 (m, 7H), 7.05-7.07 (m, 2H), 2.36 (s, 3H). LRMS m/z (M+H) calculated: 287.3, found: 287.1.

2-Chloro-6-(4-methylphenyl)-5-phenylnicotinonitrile (2-4)

Mix 6-(4-methylphenyl)-2-oxo-5-phenyl-1,2-dihydropyridine-3-carbonitrile (2-3; 1.5g, 5.24mmol) with POCl ₃ (15mL ) mixture was heated to 100°C for 3 hours and concentrated. The residue _was basified with aqueous sodium carbonate and extracted with _CH2Cl2 (3 x 30 mL). The organic layers were combined, dried, filtered and concentrated. The residue was purified by silica gel chromatography (5-10% EtOAc in hexanes) to afford 2-chloro-6-(4-methylphenyl)-5-phenylnicotinonitrile (2-4). ¹ H NMR (500MHz, CDCl ₃ ): δ7.95(s, 1H), 7.33-7.35(m, 3H), 7.28(d, J=8.3, 2H), 7.16-7.18(m, 2H), 7.06( d, J=7.9, 2H), 2.32 (s, 3H). LRMS m/z (M+H) calculated: 305.2, found: 305.1.

6-[4-(bromomethyl)phenyl]-2-chloro-5-phenylnicotinonitrile (2-5)

2-Chloro-6-(4-methylphenyl)-5-phenylnicotinonitrile (2-4; 0.066g, 0.217mmol), NBS (0.046g, 0.26mmol) and benzoyl peroxide (0.010 g, 0.043 mmol) in _CDCl3 (2 mL) was heated to reflux overnight. The resulting mixture was concentrated to afford 6-[4-(bromomethyl)phenyl]-2-chloro-5-phenylnicotinonitrile (2-5), which was used without further purification. LRMS m/z (M+H) calculated: 383.7, found: 383.1.

2-Chloro-5-phenyl-6-[4-({[4-(1,2,3-thiadiazol-4-yl)benzyl]amino}methyl)phenyl]nicotinonitrile (2- 6)

To a solution of 6-[4-(bromomethyl)phenyl]-2-chloro-5-phenylnicotinonitrile (2-5; 0.294 g, 0.766 mmol) in THF (5 mL) and MeOH (5 mL) was added 1- [4-(1,2,3-Thiadiazol-4-yl)phenyl]methanamine (0.147 g, 0.766 mmol) and DIPEA (0.495 g, 3.831 mmol). The resulting mixture was stirred overnight and concentrated. The residue was treated with _{aqueous Na2CO3} (2M, 10 mL) and extracted with _CH2Cl2 (3 _x 30 mL). The organic layers were combined, dried, filtered and concentrated. The residue was purified by silica gel chromatography (3-6% _MeOH in _CH2Cl2 ) to give 2-chloro-5-phenyl-6-[4-({[4-(1,2,3-thiadi Azol-4-yl)benzyl]amino}methyl)phenyl]nicotinonitrile (2-6). ¹ HNMR (500MHz, CDCl ₃ ): δ8.63(s, 1H), 8.01(d, J=8.0, 2H), 7.98(s, 1H), 7.47(d, J=8.1, 2H), 7.28-7.38 (m, 7H), 7.16-7.18 (m, 2H), 3.84 (s, 2H), 3.83 (s, 2H). LRMS m/z (M+H) calculated: 494.0, found: 494.1.

Compounds of Table 1 were synthesized according to Scheme 1, but substituting appropriate substituted amines for compounds (1-4) in Scheme 1: Unless otherwise indicated, the TFA salts of the indicated compounds were isolated by mass spectrometry controlled HPLC purification.

Although only one regioisomer is illustrated in Table 1, two isomers were synthesized.

Table 1

Process 3

5-Chloro-2-(4-methylphenyl)-3-phenylpyridine (3-2)

At 0°C, 1-(4-methylphenyl)-2-phenylethanone (4-1) (Renault, O; Dallemagne, P.; and Rault, S.Org.Prep.Proced.Int .1999, 31, 324) (0.5 g, 2.38 mmol) in THF (5 mL) was added to a THF solution of tert-butoxide (2.6 mL, 1 M). The resulting slurry was stirred at room temperature for 45 minutes. 2-Chloro-N,N-dimethylaminoallylidenedimethylamine hexafluorophosphate (1.1 g, 3.6 mmol) was added and the resulting mixture was heated to 45°C for 3 hours. Additional ammonium acetate (0.367 g, 4.76 mmol) was added and the resulting mixture was heated to reflux for 6 hours. The resulting solution was diluted with water and extracted with EtOAc. The organic layers were combined, dried, filtered and concentrated. The residue was purified by silica gel chromatography (2-4% EtOAc in hexanes) to afford the title compound. LRMS calculated m/z (M+H): 280.8, found 280.2.

6-(4-Methylphenyl)-5-phenylnicotinonitrile (3-3)

5-Chloro-2-(4-methylphenyl)-3-phenylpyridine (0.320g, 1.144mmol), zinc cyanide (0.081g, 0.687mmol), Pd ₂ (dba) ₃ (0.063g, A mixture of 0.069 mmol), dppf (0.076 g, 0.137 mmol) and zinc (0.018 g, 0.275 mmol) in DMA (5 mL) was degassed (3x pump/N2) and then heated to 150° C. for 20 h. The reaction was then cooled to room temperature, concentrated, partitioned between EtOAc and water, extracted 3x with EtOAc. The organic layers were combined, dried ( _{anhydrous Na2SO4} ), filtered and concentrated. Purification by silica gel chromatography (0-20% EtOAc in hexanes) afforded the title compound. ¹ H NMR (500MHz, CDCl ₃ ): δ8.92(d, 1H, J=2.0Hz), 7.95(d, 1H, J=2.2Hz), 7.34-7.33(m, 3H), 7.28-7.26(m , 2H), 7.20-7.18 (m, 2H), 7.07 (d, 2H, J=8.1 Hz), 2.33 (s, 3H) LRMS m/z (M+H) calculated: 271.0, found 271.0.

1-(2-aminophenyl)-3-({4-[5-(5-amino-1,3,4-thiadiazol-2-yl)-3-phenylpyridin-2-yl]benzyl Base}amino)propan-1-one (3-5)

To a mixture of 6-(4-methylphenyl)-5-phenylnicotinonitrile (0.250 g, 0.925 mmol) and CDCl ₃ (6 mL) were added NBS (0.173 g, 0.971 mmol) and benzoyl peroxide (0.045 g, 0.185 mmol). The reaction was heated to reflux for 15 hours. The reaction mixture was then cooled and concentrated to afford crude 6-[4-(bromomethyl)phenyl]-5-phenylnicotinonitrile. LRMS m/z (M+H) calculated: 348.9, found 348.9.

To a stirred mixture of 3-amino-1-(2-aminophenyl)propan-1-one and DIEA in 1:1 MeOH:THF (1 mL) was added crude 6-[4-(bromomethyl Base) phenyl] -5-phenylnicotinonitrile. The reaction was stirred at room temperature for 2 hours. The reaction was concentrated, taken up in DMF, and purified by reverse phase HPLC (5% _CH3CN : 95% _H2O : 0.01% TFA to 95% _CH3CN: 5% _H2O : 0.01% TFA). LRMS m/z (M+H) calcd: 433.1, found: 433.1.

A mixture of nitrile (0.014 g, 0.032 g) and thiosemicarbazide (0.004 g, 0.049 mmol) in TFA was heated to 60° C. for 15 hours. The mixture was then cooled and concentrated. The residue was taken up in DMF and purified by reverse phase HPLC (5% _CH2CN : 95% H2O _: 0.01% TFA to 95% _CH3CN : 5% H2O _: 0.01% TFA). NMR (500MHz, d ₆ DMSO) δ 12.98 (bs, 1H), 8.77 (bs, 2H), 8.08 (d, 1H, J=2.19Hz), 7.95 (d, 2H, J=7.1Hz), 7.70 ( d, 1H, J=6.84Hz), 7.67-7.60(m, 3H), 7.50(t, 2H, J=7.81Hz), 7.38-7.25(m, 7H), 6.79(d, 1H, J=7.6Hz) , 6.57(t, 1H, J=8.05Hz), 4.22(t, 2H, J=5.61Hz), 3.39(t, 2H, J=6.8Hz), 3.28-3.15(m, 2H) LRMSm/z(M +H) Calculated: 507.0, Found: 507.0.

The compounds shown in Table 2 below were prepared in a similar manner.

Table 2

Example 1

Cloning of human Akt isoforms and ΔPH-Akt1

The pS2neo vector (deposited at ATCC on April 3, 2001, accession number ATCC PTA-3253) was prepared as follows: A fragment of 2734bp was obtained. The PUChsneo vector (prepared as described in EMBO J. 4: 167-171 (1985)) was also cut with BglII and a 4029 bp band was isolated. The two isolated fragments were ligated together to generate the vector pS2neo-1. This plasmid contains a polylinker between the metallothionein promoter and the alcohol dehydrogenase plus poly A site. It also has a neo resistance gene driven by a heat shock promoter. The pS2neo-1 vector was cut with Psp5II and BsiWI. Two complementary oligonucleotides were synthesized and then annealed (CTGCGGCCGC (SEQ. ID. NO.: 1) and GTACGCGGCCGCAG (SEQ. ID. NO.: 2)). The cut pS2neo-1 and annealed oligonucleotides were ligated together to generate the second vector, pS2neo. A NotI site was added to this transformant prior to transfection of S2 cells to aid in linearization.

Human Akt1 gene uses 5' primer: 5'CGCGAATTCAGATCTACCATG-AGCGACGTGGCTATTGTG 3' (SEQ ID NO.: 3), and 3' primer: 5'CGCTCTAGAGGATCCTCAGGCCGTGCTGCTGGC3' (SEQ ID NO.: 4), obtained from human spleen by PCR (Clontech) cDNA (Clontech) amplification. The 5' primer includes EcoRI and BglII2 sites. The 3' primer includes XbaI and BamHI sites for cloning purposes. The resulting PCR product was subcloned into pGEM3Z (Promega) as an EcoRI/XbaI fragment. For expression/purification purposes, a middle T marker was added to the 5' end of the full-length Akt1 gene with PCR primer 5'GTACGATGC-TGAACGATATCTTCG 3' (SEQ. ID. NO.: 5). The resulting PCR product contains a 5'KpnI site and a 3'BamHI site for subcloning the fragment into the biotin-labeled insect cell expression vector pS2neo in the same frame.

To express Akt1 with deletion of the pleckstrin homology domain (PH) (Δaa 4-129, including deletion of part of the Akt1 hinge region), PCR deletion mutagenesis was performed using the full-length Akt1 gene in the pS2neo vector as a template . PCR was performed in two steps with the following overlapping internal primers: (5' GAATACATGCCGATGGAAAGCGACGGGGCTGAA-GAGATGGAGGTG 3' (SEQ. ID. NO.: 6) and 5' CCCCTCCATCTCTTC-AGCCCCGTCGCTTTCCATCGGCATGTATTC 3' (SEQ. ID. NO.: 7)), The overlapping internal primers contained deletions and at the 5' end contained 5' and 3' flanking primers containing a KpnI site and a middle T marker. The final PCR product was digested with Kpnl and SmaI and ligated into the pS2neo full-length Akt1 KpnI/SmaI cut vector, effectively replacing the 5' end of the clone with the deleted version.

Human Akt3 gene uses amino-terminal oligonucleotide primer: 5'GAATTCAGATCTACCATGAGCGATGTTACCATTGTG 3' (SEQ.ID.NO.: 8); and carboxy-terminal oligonucleotide primer: 5'TCTAGATCTTAT-TCTCGTCCACTTGCAGAG3' (SEQ.ID.NO. :9), amplified by PCR from adult human brain cDNA (Clontech). These primers contain 5'EcoRI/BgIII sites and 3'XbaI/BgIII sites for cloning purposes. The resulting PCR product was cloned into the EcoRI and XbaI sites of pGEM4Z (Promega). For expression/purification purposes, a middle T marker was added to the 5' end of the full-length Akt3 clone with PCR primer: 5'GGTACCATGGAATACATGCCGATGGAAAGCGATGTT-ACCATTGTGAAG 3' (SEQ.ID.NO.: 10). The resulting PCR product contains a 5' KpnI site, which allows in-frame cloning with the biotin-labeled insect cell expression vector pS2neo.

Amino-terminal oligonucleotide primers for human Akt2 gene: 5'AAGCTTAGATCTACCATGAATGAGGTGTCTGTC 3' (SEQ.ID.NO.: 11); and carboxy-terminal oligonucleotide primers: 5'GAATTCGGATCCTCACTC-GCGGATGCTGGC 3' (SEQ.ID.NO .:12), amplified by PCR from human thymus cDNA (Clontech). These primers contain 5'HindIII/BgIII sites and 3'EcoRI/BamHI sites for cloning purposes. The resulting PCR product was subcloned into the HindIII/EcoRI site of pGEM3Z (Promega). For expression/purification purposes, a middle T tag was added to the 5' end of full-length Akt2 with PCR primer: 5'GGTACCATGGAATACATGCCGATGGAAATGAGGTGTCTGTCATCAAAG 3' (SEQ. ID. NO.: 13). The resulting PCR product was subcloned into the pS2neo vector as described above.

Example 2

Expression of human Akt isoforms and ΔPH-Akt1

DNA containing cloned Akt1, Akt2, Akt3 and ΔPH-Akt1 genes in the purified pS2neo expression vector was used to transfect Drosophila S2 cells (ATCC) by the calcium phosphate method. Antibiotic (G418, 500 μg/ml) resistant cell aggregates were selected. Cells were diluted to a volume of 1.0 L (-7.0 x 10 ⁶ /ml) and biotin and CuSO ₄ were added to final concentrations of 50 μM and 50 mM, respectively. Cells were cultured at 27°C for 72 hours and harvested by centrifugation. Cell pellets were refrigerated at -70°C until use.

Example 3

Purification of human Akt isoforms and ΔPH-Akt1

Cell pellets obtained from 1 liter of S2 cells described in Example 2 were sonicated using buffer A: (50 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mMAEBSF, 10 μg/ml benzamidine, 5 μg/ml (respectively) ) leupeptin, aprotinin and pepprotin, 50ml 1% CHAPS in 10% glycerol and 1 mM DTT). The soluble fraction was purified with a protein GSepharose high flow rate (Pharmacia) column loaded with 9 mg/ml anti-T monoclonal antibody, with 75 μM EYMPME (SEQ.ID.NO.: 14 ) in buffer A containing 25% glycerol. ) peptide elution. Fractions containing Akt were pooled and the protein purity was identified by SDS-PAGE. Purified protein was quantified using the standard Bradford method. Purified proteins were snap-frozen in liquid nitrogen and stored at -70°C.

Akt and Akt pleckstrin homology domain deletions purified from S2 cells are required for activation. Akt and Akt pleckstrin homology domain deletions were cultured in the presence of 10 nM PDK1 (Upstate Biotechnology, Inc.), lipid vesicles (10 μM phosphatidylinositol-3,4,5-triphosphate-Metreya, Inc. 100 μM phosphatidylcholine and 100 μM phosphatidylserine—Avanti Polar lipids, Inc.) and activation buffer (50 mM Tris pH7.4, 1.0 mM DTT, 0.1 mM EGTA, 1.0 μM Microcystin-LR, 0.1 mM ATP, 10 mM MgCl ₂ , 333 μg/ml BSA and 0.1 mM EDTA) for activation (Alessi et al., Current Biology 7: 261-269). Reactions were incubated at 22°C for 4 hours. Aliquots of the reaction were snap frozen with liquid nitrogen.

Example 4

Akt kinase assay

Activated Akt isoforms and pleckstrin homology domain deletion constructs were analyzed using GSK-derived biotinylated peptide substrates. By using a phosphopeptide-specific lanthanide chelate (Lance)-conjugated monoclonal antibody in combination with a streptavidin (which binds to the biotin moiety of the peptide)-linked allophycocyanin (SA-APC) fluorophore, The extent of peptide phosphorylation was determined by homogeneous time-resolved fluorescence (HTRF). When Lance and APC are in close proximity (ie, bound to the same phosphopeptide molecule), nonradiative energy transfer occurs from Lance to APC, and then light is emitted from APC at a wavelength of 665 nm.

Materials required for analysis

A. Activated Akt isozyme or pleckstrin homology domain deletion construct;

B.Akt peptide substrate: GSK3α (S21) peptide no.3928 biotin-GGRARTSSFAEPG (SEQ.ID.NO.: 15), Macromolecular Resources;

C.Lance labeled anti-phospho GSK3α monoclonal antibody (Cell Signaling Technology, clone#27);

D. SA-APC (Prozyme catalog number PJ25S lot number 896067);

E. ^Microfluor® B U-shaped bottom microtiter plate (Dynex Technologies, cat. no. 7205)

F. ^Discovery® HTRF Microplate Analyzer, Packard Instrument Company;

G.100X protease inhibitor cocktail (PIC): 1mg/ml benzamidine, 0.5mg/ml pepstatin, 0.5mg/ml leupeptin, 0.5mg/ml aprotinin;

H.10X analysis buffer: 500mM HEPES, pH7.5, 1% PEG, mM EDTA, 1mM EGTA, 1% BSA, 20mM θ-glycerophosphate;

I. Quenching buffer: 50mM HEPES pH7.3, 16.6mM EDTA, 0.1%BSA, 0.1%Triton-100, 0.17nM Lance labeled monoclonal antibody clone #27, 0.0067mg/ml SA-APC;

J. ATP/MgCl ₂ working solution: 1X assay buffer, 1mM DTT, 1X PIC, 125mM KCl, 5% glycerol, 25mM MgCl ₂ , 375TM ATP;

K. Enzyme working solution: 1X assay buffer, 1mM DTT, 1X PIC, 5% glycerol, active Ak. Choose the final concentration of the enzyme so that the assay is performed within the linear response range;

L. Peptide working solution: 1X assay buffer, 1 mM DTT, 1X PIC, 5% glycerol, 2TMGSK3 biotinylated peptide #3928.

Add 16TL of ATP/MgCl ₂ working solution to the appropriate wells of the 96-well microtiter plate to assemble the reaction system. Add inhibitor or vehicle (1.0Tl), followed by 10Tl peptide working solution. The reaction was started by adding 13Tl enzyme working solution and mixing. The reaction was allowed to proceed for 50 minutes and then stopped by adding 60 Tl of HTRF quenching buffer. Terminated reactions were incubated at room temperature for at least 30 minutes before being read on the Discovery instrument.

PKA analysis:

Each independent PKA assay consisted of the following components:

A.5X PKA analysis buffer (200mM Tris pH7.5, 100mM MgCl ₂ , 5mMθ-mercaptoethanol, 0.5mM EDTA);

B.50 μM Kemptide (Sigma) stock solution, diluted with water;

C. ³³ P-ATP, prepared by diluting 1.0 μl ³³ P-ATP [10 mCi/ml] into 200 Tl of 50 μM unlabeled ATP stock solution;

D. 10 μl 70nM PKA catalytic subunit (UBI catalog number 14-114) stock solution, diluted with 0.5mg/ml BSA;

E. PKA/kemptide working solution: equal volume of 5X PKA assay buffer, kemptide solution and PKA catalytic subunit.

Assemble the reaction system in a 96-deep well assay plate. Inhibitor or vehicle (1.0 Tl) was added to 10 Tl of ³³ P-ATP solution and the reaction was initiated by adding 30 Tl of PKA/kemptide working solution to each well. The reactions were mixed and incubated at room temperature for 20 minutes. The reaction was stopped by adding 50Tl of 100 mM EDTA and 100 mM sodium pyrophosphate with mixing.

Enzyme reaction products (phosphorylated kemptide) were collected on p81 phosphocellulose 96-well filter plates (Millipore). Filter plates were prepared by filling wells of p81 filter plates with 75 mM phosphoric acid. The wells can be evacuated from the filter plate by applying a vacuum at the bottom of the filter plate. Phosphoric acid (75 mM, 170 μl) was added to each well. Aliquots of 30 μl from each PKA reaction that had been terminated were added to the corresponding wells on the filter plates containing phosphoric acid. After vacuum was applied, the peptides were trapped on the filter plates, which were washed 5 times with 75 mM phosphoric acid. After the last wash, allow the filter plates to air dry. Scintillation fluid (30 [mu]l) was added to each well and the filter plates were counted with a TopCount (Packard).

PKC analysis:

Each PKC assay consists of the following components:

A.10X PKC co-activation buffer: 2.5mM EGTA, 4mM CaCl ₂ ;

B.5X PKC activation buffer: 1.6mg/ml phosphatidylserine, 0.16mg/ml diacylglycerol, 100mM Tris pH7.5, 50mM MgCl ₂ , 5mMθ-mercaptoethanol;

C. ³³ P-ATP, prepared by diluting 1.0 μl ³³ P-ATP [10 mCi/ml] into 100 μl of 100 μM unlabeled ATP stock solution;

D. Myelin basic protein (350 μ g/ml, UBI), diluted with water;

E. PKC (50ng/ml, UBI catalog number 14-115), diluted with 0.5mg/ml BSA;

F. PKC/MBP working solution: prepared by mixing 5 volumes each of PKC coactivation buffer and MBP with 10 volumes each of PKC activation buffer and PKC.

Assemble the assay system in a 96-deep well assay plate. Inhibitor or vehicle (1.0 Tl) was added to 5.0 ^ul33P -ATP. Reactions were initiated by adding PKC/MBP working solution and mixing. The reaction solution was incubated at 30°C for 20 minutes. The reaction was stopped by adding 50 Tl of 100 mM EDTA and 100 mM sodium pyrophosphate with mixing. Phosphorylated myelin basic protein was collected onto PVDF membranes in 96-well filter plates and quantified by scintillation counting.

Specific compounds of the invention were tested in the assays described above and found to have an IC50 ≤ ₅₀ μM for one or more of Akt1, Akt2 and Akt3.

Example 5

Cell-Based Assays to Measure Akt Inhibition

Cells (such as LnCap or PTEN ^(-/-) tumor cell lines with activated Akt) were plated in 100 mM dishes. When the cells are approximately 70-80% confluent on the plate, the cells are re-cultivated with 5 ml of fresh medium, and the compound to be tested is added to the solution. Controls included untreated cells, vehicle-treated cells and cells treated with 20 [mu]M or 200 nM LY294002 (Sigma) or wortmannin (Sigma), respectively. Cells were incubated for 2, 4 or 6 hours and then the medium was removed. Cells were washed with PBS, scraped and transferred to centrifuge tubes. The cells were centrifuged to obtain a pellet, which was washed again with PBS. Finally, the cell pellet was resuspended in lysis buffer (20mM Tris pH8, 140mM NaCl, 2mM EDTA, 1% Triton, 1mM sodium pyrophosphate, 10mM θ-glycerophosphate, 10mM NaF, 0.5mmNaVO ₄ , 1μM Microsystine and 1x protease inhibitor mixture), placed on ice for 15 minutes and swirled gently to lyse the cells. The lysate was spun at 100,000 xg in a Beckman benchtop ultracentrifuge for 20 minutes at 4°C. Supernatant proteins were quantified by the standard Bradford method (BioRad) and stored at -70°C until use.

Proteins were immunoprecipitated (IP) from clarified lysates as follows: For Akt1, lysates were mixed with NETN from Santa Cruz sc-7126 (D-17) (100 mM NaCl, 20 mM Tris pH8.0, 1 mM EDTA, 0.5% NP-40) The solution was mixed and protein A/G agarose (Santa Cruz sc-2003) was added. For Akt2, the lysate was mixed in NETN with anti-Akt-2 agarose (Upstate Biotechnology #16-174) and for Akt3, the lysate was mixed in NETN with anti-Akt3 agarose (Upstate Biotechnology #16-175). Immunoprecipitates were incubated overnight at 4°C, washed, and separated by SDS-PAGE.

Western blot was used to analyze the corresponding phosphorylation sites on total Akt, pThr308 Akt1, pSer473 Akt1 and Akt2 and Akt3, and specific antibodies (Cell Signaling Technology): anti-total Akt (catalogue number 9272), anti-phospho-Akt serine 473 ( Cat. No. 9271) and Anti-Phospho-Akt Threonine 308 (Cat. No. 9275), for analysis of downstream targets of Akt. The blot was incubated overnight at 4°C with the appropriate primary antibody diluted in PBS+0.5% nonfat dry milk (NFDM), washed, and incubated with horseradish peroxidase (HRP) diluted in PBS+0.5% NFDM at room temperature Labeled secondary antibody was incubated for 1 hour. Proteins were detected with ECL reagents (Amersham/Pharmacia Biotech RPN2134).

Example 6

Heregulin-stimulated Akt activation

MCF7 cells (human breast cancer cell line, PTEN ^+/+ ) were plated at a concentration of 1×10 ⁶ cells per 100 mM plate. When the cells are about 70-80% of the plate, add 5ml of serum-free medium and incubate overnight. The next morning, compounds were added, cells were incubated for 1-2 hours, then heregulin was added over 30 minutes (to induce activation of Akt), and cells were analyzed as described above.

Example 7

inhibition of tumor growth

The in vivo efficacy of cancer cell growth inhibitors can be confirmed by several methods well known in the art.

On day 0, human tumor cell lines with dysregulated PI3K pathway (such as LnCaP, PC3, C33a, OVCAR-3, MDA-MB-468, etc.) were subcutaneously injected into the left flank of 6-10-week-old female nude mice (Harlan). . Nude mice are randomly assigned to vehicle, compound or combination treatment groups. From day 1, subcutaneous administration was carried out daily and continued throughout the experimental process. Alternatively, the inhibitor compound to be tested can be administered by a continuous infusion pump. The total volume of compound, compound combination or vehicle administered is 0.2 ml. When lesions with a diameter of 0.5-1.0 cm appeared in all vehicle-treated nude mice, usually 4-5.5 weeks after cell injection, the tumors were excised and weighed. The average weight of tumors in each experimental group for each cell line was calculated.

Sequence Listing

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Duggan, Mark E.

Hartnett, John C.

Lindsley, Craig W.

Wu, Zhicai

Zhao, Zhijian

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Claims (18)

1. A compound represented by formula A or its pharmaceutically acceptable salt or stereoisomer:

in: a is 0 or 1; b is 0 or 1; m is 0, 1 or 2; n is 0, 1, 2 or 3; p is 0, 1 or 2; q is 0, 1, 2 or 3; r is 0 or 1; s is 0 or 1; t is 2, 3, 4, 5 or 6; R ¹ is independently selected from 1) (C=O) _a O _b C ₁ -C ₁₀ alkyl, 2) (C=O) _a O _b aryl, 3) C ₂ -C ₁₀ alkenyl, 4) C ₂ -C ₁₀ alkynyl, 5) (C=O) _a O _b heterocyclyl, 6) (C=O) _a O _b C ₃ -C ₈ cycloalkyl, 7) CO ₂ H, 8) halogen, 9 ) CN, 10) OH, 11) O _b C ₁ -C ₆ perfluoroalkyl, 12) O _a (C=O) _b NR ⁶ R ⁷ , 13) NR ^c (C=O) NR ⁶ R ⁷ , 14) S(O) _m R ^a , 15) S(O) ₂ NR ⁶ R ⁷ , 16) NR ^c S(O) _m R ^a , 17) oxo, 18) CHO, 19) NO ₂ , 20) NR ^c (C=O)O _b R ^a , 21)O(C=O)O _b C ₁ -C ₁₀ alkyl, 22)O(C=O)O _b C ₃ -C ₈ cycloalkyl, 23 )O(C=O)O _b aryl, 24)O(C=O)O _b -heterocycle and 25)O _a -P=O(OH) ₂ , the alkyl, aryl, alkenyl, Alkynyl, heterocyclyl and cycloalkyl are optionally substituted by one or more substituents selected from ^Rz ; R ² is independently selected from 1) (C=O) _a O _b C ₁ -C ₁₀ alkyl, 2) (C=O) _a O _b aryl, 3) C ₂ -C ₁₀ alkenyl, 4) C ₂ -C ₁₀ alkynyl, 5) (C=O) _a O _b heterocyclyl, 6) (C=O) _a O _b C ₃ -C ₈ cycloalkyl, 7) CO ₂ H, 8) halogen, 9 ) CN, 10) OH, 11) O _b C ₁ -C ₆ perfluoroalkyl, 12) O _a (C=O) _b NR ⁶ R ⁷ , 13) NR ^c (C=O) NR ⁶ R ⁷ , 14) S(O) _m R ^a , 15) S(O) ₂ NR ⁶ R ⁷ , 16) NR ^c S(O) _m R ^a , 17) CHO, 18) NO ₂ , 19) NR ^c (C= O)O _b R ^a , 20)O(C=O) _Ob C ₁ -C ₁₀ alkyl, 21)O(C=O)O _b C ₃ -C _8cycloalkyl , 22)O(C= O)O _b aryl, 23) O(C=O)O _b -heterocycle and 24)O _a -P=O(OH) ₂ , the alkyl, aryl, alkenyl, alkynyl, heterocycle and cycloalkyl are optionally substituted by one, two or three substituents selected from R ^z ; R ³ and R ⁴ are independently selected from H, C ₁ -C ₆ -alkyl and C ₁ -C ₆ -perfluoroalkyl, or R ³ and R ⁴ are combined to form -(CH ₂ ) _t -, one of which A carbon atom is optionally replaced by a moiety selected from O, S(O) _m , -N( ^Rb )C(O)- and -N( ^CORa )-; R ⁵ is independently selected from 1) (C=O) _a O _b C ₁ -C ₁₀ alkyl, 2) (C=O) _a O _b aryl, 3) C ₂ -C ₁₀ alkenyl, 4) C ₂ -C ₁₀ alkynyl, 5) (C=O) _a O _b heterocyclyl, 6) (C=O) _a O _b C ₃ -C ₈ cycloalkyl, 7) CO ₂ H, 8) halogen, 9 ) CN, 10) OH, 11) O _b C ₁ -C ₆ perfluoroalkyl, 12) O _a (C=O) _b NR ⁶ R ⁷ , 13) NR ^c (C=O) NR ⁶ R ⁷ , 14) S(O) _m R ^a , 15) S(O) ₂ NR ⁶ R ⁷ , 16) NR ^c S(O) _m R ^a , 17) oxo, 18) CHO, 19) NO ₂ , 20) O(C=O)O _b C ₁ -C ₁₀ alkyl, 21)O(C=O)O _b C ₃ -C ₈ cycloalkyl and 22)O _a -P=O(OH) ₂ , the Alkyl, aryl, alkenyl, alkynyl, heterocyclyl and cycloalkyl are optionally substituted by one or more substituents selected from ^R ; R ⁶ and R ⁷ are independently selected from 1) H, 2) (C=O)O _b R ^a , 3) C ₁ -C ₁₀ alkyl, 4) aryl, 5) C ₂ -C ₁₀ alkenyl, 6 ) C ₂ -C ₁₀ alkynyl, 7) heterocyclyl, 8) C ₃ -C ₈ cycloalkyl, 9) SO ₂ R ^a , 10) (C=O)NR ^b ₂ , 11) OH and 12) O _a -P=O(OH) ₂ , the alkyl, cycloalkyl, aryl, heterocyclyl, alkenyl and alkynyl are optionally substituted by one or more substituents selected from R ^z ; R ^z is selected from 1) (C=O) _r O _s (C ₁ -C ₁₀ ) alkyl, 2) O _r (C ₁ -C ₃ ) perfluoroalkyl, 3) (C ₀ -C ₆ ) alkylene Alkyl-S(O) _m R ^a , 4) oxo, 5) OH, 6) halogen, 7) CN, 8) (C=O) _r O _s (C ₂ -C ₁₀ ) alkenyl, 9) (C=O) _rOs ( _C2 - _C10 )alkynyl, 10)(C= _O ) _rOs ( _C3 - _C6 ) _cycloalkyl , 11)(C=O) _{rOs (} C ₀ -C ₆ )alkylene-aryl, 12)(C ₌ O) _rOs (C ₀ -C ₆ )alkylene-heterocyclyl, ₁₃ )(C=O) _rOs (C ₀ -C ₆ )alkylene-N(R ^b ) ₂ , 14)C(O)R ^a , 15)(C ₀ -C ₆ )alkylene-CO ₂ R ^a , 16)C(O)H , 17) (C ₀ -C ₆ ) alkylene-CO ₂ H, 18) C(O)N(R ^b ) ₂ , 19) S(O) _m R ^a , 20) S(O) ₂ N( R ^b ) ₂ , 21)NR ^c (C=O)O _b R ^a , 22)O(C=O)O _b C ₁ -C ₁₀ alkyl, 23)O(C=O)O _b C ₃ - C ₈ cycloalkyl, 24)O(C=O)O _b aryl, 25)O(C=O)O _b -heterocycle and 26)O _a -P=O(OH) ₂ , the alkyl , alkenyl, alkynyl, cycloalkyl, aryl and heterocyclyl are optionally replaced by up to three selected from R ^b , OH, (C ₁ -C ₆ )alkoxy, halogen, CO ₂ H, CN, O Substituents of (C=O)C ₁ -C ₆ alkyl, oxo, N(R ^b ) ₂ and O _a -P=O(OH) ₂ ; R ^a is: substituted or unsubstituted (C ₁ -C ₆ ) alkyl, substituted or unsubstituted (C ₂ -C ₆ ) alkenyl, substituted or unsubstituted (C ₂ -C ₆ ) alkynyl, substituted or unsubstituted (C ₃ -C ₆ ) cycloalkyl, substituted or unsubstituted aryl, (C ₁ -C ₆ ) perfluoroalkyl, 2,2,2-trifluoroethyl or substituted or unsubstituted the heterocyclic group; R ^b is: H, (C ₁ -C ₆ )alkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted heterocyclyl, (C ₃ -C ₆ )cycloalkane radical, (C=O)OC ₁ -C ₆ alkyl, (C=O)C ₁ -C ₆ alkyl or S(O) ₂ R ^a ; R ^c is selected from 1) H, 2) C ₁ -C ₁₀ alkyl, 3) aryl, 4) C ₂ -C ₁₀ alkenyl, 5) C ₂ -C ₁₀ alkynyl, 6) heterocyclyl, 7 ) C ₃ -C ₈ cycloalkyl and 8) C ₁ -C ₆ perfluoroalkyl, said alkyl, cycloalkyl, aryl, heterocyclyl, alkenyl and alkynyl are optionally replaced by one or more Substituents selected from ^Rz are substituted. 2. The compound of claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, said compound being of formula B:

in: Rz ^' is selected from the group consisting of: alkyl, cycloalkyl, aryl and heterocyclyl optionally substituted with 1-3 ^Rz . 3. The compound of claim 2 or a pharmaceutically acceptable salt or stereoisomer thereof, said compound being of formula C:

in: R ⁶ is selected from: H and (C ₁ -C ₆ )alkyl. 4. A compound selected from the group consisting of: 5-phenyl-6-[4-({[4-(1,2,3-thiadiazol-4-yl)benzyl]amino}methyl)phenyl]nicotinonitrile; 5-Phenyl-6-[4-({[(1S,2R)-2-phenylcyclopropyl]amino}methyl)phenyl]nicotinonitrile; 6-(4-{[(3,4-difluorobenzyl)amino]methyl}phenyl)-5-phenylnicotinonitrile; 6-[4-({[2-(3-fluorophenyl)ethyl]amino}methyl)phenyl]-5-phenylnicotinonitrile; 6-[4-({[2-(4-fluorophenyl)ethyl]amino}methyl)phenyl]-5-phenylnicotinonitrile; 5-phenyl-6-[4-({[(4-phenylmorpholin-2-yl)methyl]amino}methyl)phenyl]nicotinonitrile; 6-[4-({[(4-Benzylmorpholin-2-yl)methyl]amino}methyl)phenyl]-5-phenylnicotinonitrile; 6-[4-({Methyl[(1-phenyl-1H-pyrazol-4-yl)methyl]amino}methyl)phenyl]-5-phenylnicotinonitrile; N-[2-(1-methylpyrrolidin-2-yl)ethyl]-N-{4-[3-phenyl-5-(1H-tetrazol-5-yl)pyridin-2-yl] Benzyl}amine; 1-{4-[3-phenyl-5-(1H-tetrazol-5-yl)pyridin-2-yl]phenyl}-N-[4-(1,2,3-thiadiazole-4 -yl)benzyl]methylamine; N-(3,4-difluorobenzyl)-N-{4-[3-phenyl-5-(1H-tetrazol-5-yl)pyridin-2-yl]benzyl}amine; 2-Chloro-5-phenyl-6-[4-({[4-(1,2,3-thiadiazol-4-yl)benzyl]amino}methyl)phenyl]nicotinonitrile; 1-(2-aminophenyl)-3-({4-[5-(5-amino-1,3,4-thiadiazol-2-yl)-3-phenylpyridin-2-yl]benzyl Base} amino) propan-1-one; 3-({4-[5-cyano-3-phenylpyridin-2-yl]benzyl}amino)-1-phenylpropan-1-one; 3-({4-[5-(5-amino-1,3,4-thiadiazol-2-yl)-3-phenylpyridin-2-yl]benzyl}amino)-1-phenylpropane -1-one; or a pharmaceutically acceptable salt or stereoisomer thereof. 5. The TFA salt of the compound of claim 1, said compound being: 5-phenyl-6-[4-({[4-(1,2,3-thiadiazol-4-yl)benzyl]amino}methyl)phenyl]nicotinonitrile; 5-Phenyl-6-[4-({[(1S,2R)-2-phenylcyclopropyl]amino}methyl)phenyl]nicotinonitrile; 6-(4-{[(3,4-difluorobenzyl)amino]methyl}phenyl)-5-phenylnicotinonitrile; 6-[4-({[2-(3-fluorophenyl)ethyl]amino}methyl)phenyl]-5-phenylnicotinonitrile; 6-[4-({[2-(4-fluorophenyl)ethyl]amino}methyl)phenyl]-5-phenylnicotinonitrile; 5-phenyl-6-[4-({[(4-phenylmorpholin-2-yl)methyl]amino}methyl)phenyl]nicotinonitrile; 6-[4-({[(4-Benzylmorpholin-2-yl)methyl]amino}methyl)phenyl]-5-phenylnicotinonitrile; 6-[4-({Methyl[(1-phenyl-1H-pyrazol-4-yl)methyl]amino}methyl)phenyl]-5-phenylnicotinonitrile; N-[2-(1-methylpyrrolidin-2-yl)ethyl]-N-{4-[3-phenyl-5-(1H-tetrazol-5-yl)pyridin-2-yl] Benzyl}amine; 1-{4-[3-phenyl-5-(1H-tetrazol-5-yl)pyridin-2-yl]phenyl}-N-[4-(1,2,3-thiadiazole-4 -yl)benzyl]methylamine; N-(3,4-difluorobenzyl)-N-{4-[3-phenyl-5-(1H-tetrazol-5-yl)pyridin-2-yl]benzyl}amine; 1-(2-aminophenyl)-3-({4-[5-(5-amino-1,3,4-thiadiazol-2-yl)-3-phenylpyridin-2-yl]benzyl Base} amino) propan-1-one; 3-({4-[5-(5-amino-1,3,4-thiadiazol-2-yl)-3-phenylpyridin-2-yl]benzyl}amino)-1-phenylpropane -1-one; or its stereoisomers. 6. The compound of claim 4, which is: 1-(2-aminophenyl)-3-({4-[5-(5-amino-1,3,4-thiadiazol-2-yl)-3-phenylpyridin-2-yl]benzyl Base} amino) propan-1-one; or a pharmaceutically acceptable salt or stereoisomer thereof. 7. A pharmaceutical composition comprising a pharmaceutical carrier and a therapeutically effective amount of the compound of claim 1 dispersed in the carrier. 8. A pharmaceutical composition comprising a pharmaceutical carrier and a therapeutically effective amount of the compound of claim 4 dispersed in the carrier. 9. A method of inhibiting one or more isoforms of Akt in a mammal, said method comprising administering to the mammal a therapeutically effective amount of a compound of claim 1. 10. A method of inhibiting one or more isoforms of Akt in a mammal, said method comprising administering to the mammal a therapeutically effective amount of a compound of claim 4. 11. A method of treating cancer comprising administering a therapeutically effective amount of a compound of claim 1 to a mammal in need thereof. 12. A method of treating cancer comprising administering to a mammal in need thereof a therapeutically effective amount of a compound of claim 4. 13. A method of treating a non-malignant disease involving angiogenesis, said method comprising administering a therapeutically effective amount of a compound of claim 1 to a mammal in need thereof. 14. A method of treating a non-malignant disease involving angiogenesis, said method comprising administering a therapeutically effective amount of a compound of claim 4 to a mammal in need thereof. 15. The composition of claim 8, further comprising a second compound selected from the group consisting of 1) an estrogen receptor modulator, 2) an androgen receptor modulator, 3) a retinoid receptor Modulators, 4) Cytotoxic/cytostatic agents, 5) Antiproliferative agents, 6) Prenyl protein transferase inhibitors, 7) HMG-CoA reductase inhibitors, 8) HIV protease inhibitors, 9) Reverse transcriptase inhibitors, 10) angiogenesis inhibitors, 11) PPAR-gamma agonists, 12) PPAR-delta agonists, 13) inhibitors of cell proliferation and survival signaling, 14) drugs that interfere with cell cycle checkpoints. 16. A method of treating cancer comprising administering a therapeutically effective amount of a compound of claim 1 in combination with radiation therapy. 17. A method of treating a proliferative disorder selected from the group consisting of restenosis, inflammation, autoimmune disease and allergy/asthma, said method comprising administering a therapeutically effective amount of a compound of claim 1 to a mammal in need thereof. 18. A method of treating insulin hypersecretion comprising administering a therapeutically effective amount of a compound of claim 1 to a mammal in need thereof.